METHODS  OP 
ORGANIC   ANALYSIS 


THE  MACMILLAN  COMPANY 

NEW  YORK  •    BOSTON  •    CHICAGO 
DALLAS    •    SAN   FRANCISCO 

MACMILLAN  &  CO.,  LIMITED 

LONDON   •    BOMBAY  •    CALCUTTA 
MELBOURNE 

THE  MACMILLAN  CO.  OF  CANADA,  LTD. 

TORONTO 


METHODS    OF 


ORGANIC    ANALYSIS 


BY 


HENRY  C.   SHERMAN,  PH.D. 

PROFESSOR   OF   FOOD   CHEMISTRY   IN   COLUMBIA   UNIVERSITY 
AUTHOR    OF    "  CHEMISTRY  OF  FOOD   AND   NUTRITION  " 


SECOND  EDITION 
REWRITTEN  AND  ENLARGED 


gork 

THE  MACMILLAN  COMPANY 
1912 

All  rights  reserved 


!  e,r' 


COPYRIGHT,  1905, 
BY  HENRY  C.   SHERMAN. 

COPYRIGHT,  1912, 
BY  THE  MACMILLAN  COMPANY. 

Set  up  and  electrotyped.    Published  June,  1912. 


Norfaooi 

J.  8.  Gushing  Co.  —  Berwick  &  Smith  Co. 
Norwood,  Mass.,  U.S.A. 


PREFACE  TO  SECOND  EDITION 

IN  rewriting  this  work  after  six  years  of  constant  use  in  the 
classroom  and  laboratory,  the  author  has  endeavored  to  keep 
in  mind  the  needs  both  of  students  and  of  practising  chemists. 
Methods  which  are  commonly  used  as  exercises  for  beginners 
(such,  for  example,  as  the  determination  of  alcohol  by  the 
distillation  method)  are  fully  described  with  detailed  explana- 
tory and  precautionary  notes,  while  those  methods  which  are 
apt  to  be  used  only  by  advanced  students  or  professional 
chemists  are  given  more  concisely.  At  the  end  of  each 
chapter  will  be  found,  first  a  list  of  reference  books  arranged 
alphabetically  by  authors,  and  then  a  chronological  list  of 
journal  articles,  bulletins,  etc.,  particularly  of  the  last  few 
years.  The  abbreviations  are  those  used  by  the  American 
Chemical  Society  in  the  publication  of  Chemical  Abstracts. 

The  scope  of  the  work  has  been  somewhat  extended,  the 
new  matter  including  a  chapter  on  solid  and  liquid  fuels,  and 
sections  on  industrial  alcohol,  drying  oils,  crude  petroleum, 
the  new  international  methods  of  glycerin  analysis,  and  quan- 
titative methods  for  the  testing  of  enzymes.  The  discussions 
of  aldehydes,  sugars,  proteins,  and  food  preservatives  are  also 
much  fuller  than  in  the  first  edition. 

These  additions,  and  the  rewriting  of  the  original  text  to 
embody  recent  advances  and  certain  changes  of  arrangement 
which  have  been  found  advantageous  from  the  standpoint  of 
teaching,  make  the  present  edition  practically  a  new  work. 

The  text  and  references  are  designed  to  cover,  along  with 
the  directions  for  laboratory  work,  so  much  at  least  of  the 
technology  of  the  various  topics  considered  as  is  involved 
in  a  proper  appreciation  of  the  purposes  of  the  analyses  and 
the  significance  of  the  analytical  results. 

258727 


VI  PREFACE    TO    SECOND    EDITION 

The  writer  takes  pleasure  in  acknowledging  his  indebtedness 
to  Dr.  C.  A.  Browne,  Chemist-in-charge  of  the  New  York 
Sugar  Trade  Laboratory,  and  Mr.  T.  T.  Gray,  Chief  Chemist 
of  the  Tide-Water  Oil  Company,  for  criticism  of  the  sections 
relating  to  their  specialties,  and  to  his  colleague,  Dr.  J.  M. 

Nelson,  for  many  helpful  suggestions. 

H.  C.  S. 
JANUARY,  1912. 


PREFACE  TO   FIRST  EDITION 

THE  purpose  of  this  work  is  to  give  a  connected  introductory 
training  in  organic  analysis,  especially  as  applied  to  plant  and 
animal  substances  and  their  manufactured  products.  No  attempt 
is  made  to  touch  upon  all  important  branches  of  this  subject, 
but  representative  topics  are  treated  in  considerable  detail 
with  reference  both  to  analytical  methods  and  to  the  interpre- 
tation of  results. 

The  greater  part  of  the  book  is  devoted  to  quantitative 
methods  for  food  materials  and  related  substances.  Standard 
works  of  reference  and  the  publications  of  the  Association  of 
Official  Agricultural  Chemists  have  been  freely  used.  The 
nomenclature  adopted  in  these  publications  has  been  followed 
as  closely  as  possible.  As  a  rule,  footnotes  show  the  original 
sources  of  statements  or  methods  included  in  the  text,  while 
general  or  additional  references  are  given  at  the  end  of  each 
chapter.  The  references  have  been  carefully  selected  and  are 
believed  to  be  sufficient  to  put  the  reader  in  touch  with  the 
most  important  literature. 

The  descriptions  of  methods  were  written  primarily  for  the 
use  of  third-year  students  in  the  School  of  Chemistry,  Colum- 
bia University,  and  therefore  presuppose  a  knowledge  of  inor- 
ganic quantitative  analysis,  elementary  organic  chemistry,  and 
general  physics. 

The  writer  takes  pleasure  in  acknowledging  his  indebtedness 
to  Professor  Edmund  H.  Miller  for  helpful  advice  and  sugges- 
tions throughout  the  work,  and  to  Mr.  Roland  H.  Williams 
for  assistance  in  testing  methods  and  in  the  revision  of  parts 
of  the  manuscript. 

H.  C.   S. 
NEW  YORK,  July  1,  1905. 

vii 


CONTENTS 

\ 


PACK 

INTRODUCTION xv 


CHAPTER  I 

ALCOHOLS 1 

Ethyl  Alcohol 2 

Detection  and  Identification  of  Ethyl  Alcohol          ...  5 

Determination  of  Ethyl  Alcohol 6 

Determination  and  Identification  of  Small  Amounts  of  Alcohol  23 

Detection  and  Determination  of  Methyl  Alcohol      ...  24 

Determination  of  Amyl  Alcohols  or  Fusel  Oil          ...  28 

Official  Requirements  of  Purity 29 

References 31 

CHAPTER  II 

ALDEHYDES     ............  34 

Formaldehyde ,         .         .  36 

Detection  and  Identification      .......  38 

Quantitative  Determination 40 

Benzaldehyde 46 

Vanillin 48 

References 48 

CHAPTER  III 

CARBOHYDRATES  —  GENERAL  METHODS 50 

Occurrence  and  Relations         » ,50 

Solubilities        ...        .        .    " .54 

Reactions  with  Acids        .........  56 

Reactions  with  Hydrazines 61 

Reduction  of  Copper  Solutions .         .  69 

Rotation  of  Polarized  Light     *        .         .        .        .     .  .        .         .78 

References         . .  85 

ix 


CONTENTS 


CHAPTER  IV 

PAGB 

SPECIAL  METHODS  OF  SUGAR  ANALYSIS     .        .        .        .  .87 

Analysis  of  Raw  Sugar     .........       87 

Polariscopic  Examination          .......       87 

Determination  of  Reducing  Sugars  .         .        .        .        .        .96 

Determination  of  Moisture  and  Ash          .        .         .        .         .97 

Determination~of  Sucrose  in  Beets  and  Cane  .        .  .         .98 

Density  and  Purity  of  Sugar  Solutions 100 

Identification  and  Analysis  of  "  Unknown  "  Sugars         .         .         .     101 
References '  ...     103 

CHAPTER  V 

STARCH  AND  AMYLASE 106 

Determination  of  Starch  .         .        .         .        .  /  .  .     106 

Diastatic  Power  of  Amylases   .         .         .         .         .         ..'.113 

References         .        .         .         .        .        .        .         .        .        .        .     121 

CHAPTER  VI 

VINEGAR  AND  ACETATE *   ;.  .     123 

Vinegar     .         .         .         .         .         .         .         .         .        .-        .  .123 

Determination  of  Source  .         .         .        .        .        .  .     124 

Methods  of  Analysis .         .         .         .         .        .        .        .  .     127 

Acetic  Acid  and  Acetates .  .     129 

References .     131 

CHAPTER  VII 

FATTY  ACIDS .133 

Acids  of  Series  C;H2nO2 .  .133 

Acids  of  Series  CnH2n_2O2 .  .     135 

Acids  of  Series  CwH2n_4O2 ",..'.     136. 

Acids  of  Series  CnH2n_6O2 '      .  .     137 

Acids  of  Series  CnH2n_8O2 .  '       .  .137 

Saturated  Hydroxy  Acids .137 

Hydroxy  Acids  of  Series  CnIl2n_2O3         .        .         .        .        .  .138 

References .  .139 

CHAPTER  VIII 

OILS,  FATS,  AND  WAXES  —  GENERAL  METHODS          ....     140 

Classification 141 

Properties  of  Fats  and  Fatty  Oils     ......  142 


CONTENTS  XI 

PAGE 

Analytical  Methods 143 

Saponification  Number 144 

Acid  and  Ester  Numbers 147 

Hehner  Number 148 

Reichert-Meissl  Number .         .  148 

Iodine  Numbers         .         .         .         .         .         .         .         .         .  148 

Maumene  Number —  Specific  Temperature  Reaction       .         .  157 

Acetyl  Number 160 

Specific  Gravity 162 

Index  of  Refraction  .         .         .         .         .         .         .         .         .164 

Alcohols  of  Fats  and  Waxes — Unsaponifiable  Matter    .        .        .  169 

References 172 

CHAPTER  IX 

EDIBLE  OILS  AND  FATS         .........  174 

Salad  Oils .174 

Analytical  Properties  of  Olive  Oil 175 

Detection  of  Adulterants 177 

Butter        .                 185 

Determination  of  Water,  Fat,  Curd,  Ash          .         .         .         .  186 

Examination  of  Butter  Fat 188 

References .         .  199 

CHAPTER  X 

DRYING  OILS 203 

Oils  Used  as  Drying  Oils 203 

Analytical  Properties  of  Linseed  Oil       .......  205 

Adulterants  and  Methods  of  Detection    ......  206 

Oils  Altered  by  Age  or  Oxidation 210 

"  Unknown "  Oils  and  Mixtures       .                  213 

References 214 

CHAPTER  XI 

PETROLEUM  AND  LUBRICATING  OILS 218 

Examination  of.  Crude  Petroleum 218 

Examination  of  Lubricating  Oils     .         .         .         .         .         .         .  222 

Determination  of  Constituents 223 

Viscosity 226 

Acidity 231 

Cold  Test  and  Chilling  Point  or  Cloud  Test     .         .        .         .  232 

Flashing  and  Burning  Points .  233 

Additional  Determinations        .......  234 

Examination  of  Lubricating  Greases 235 

References         .         .         .  236 


Xll  CONTENTS 


CHAPTER   XII 

PAGE 

FUELS 239 

Determination  of  Calorific  Power    .         .         .        .                 .         .  239 

Chemical  Composition  and  Calorific  Power  of  Organic  Compounds  245 

Fuel  Oils  and  Gasoline 247 

Woods  and  Similar  Fuels         .         .         ...        .         .         .251 

Coal 254 

Ultimate  Composition  and  Calorific  Power      ....  254 

Proximate  Analysis .        .        .         .  256 

Relation  of  Proximate  Composition  to  Calorific  Power   .         .  257 

References  261 


CHAPTER  XIII 

SOAP  AND  GLYCERIN 266 

Analysis  of  Commercial  Soap 266 

Scheme  of  Analysis 266 

Details  of  Determinations  .  .  .  .  .  267 

Glycerol  ....'....,*...  275 

Analysis  of  Crude  Glycerin  .  .  .  .  ....  276 

References  ,  286 


CHAPTER  XIV 

NITROGEN,  SULPHUR,  AND  PHOSPHORUS      .        .  .        .        .  288 

Determination  of  Nitrogen       ........  288 

Determination  of  Sulphur         .         .         .         .         .        •         .         .  295 

Determination  of  Phosphorus  ........  303 

References         ...........  306 


CHAPTER  XV 

PROTEINS  AND  PROTEASES     .        .        .        .        ...        .        .  308 

Analytical  Reactions  of  Proteins      .         .         ...         .         .  313 

Color  Reactions          .........  313 

Protein  Precipitants  .         .         .         •         •         •         .   •      .         .  316 
Separation  of  Proteins  from  Simpler  Nitrogen  Compounds  and 

from  each  other     .        .        .         .        .         •         •  •         •  322 

Proteases  or  Proteolytic  Enzymes    .         .        .  .        .  323 

References         .         .         .         .         .         •         •         •         •        *         •  329 


CONTENTS  xiii 


CHAPTER  XVI 

PAGE 

GRAIN  PRODUCTS   .        .        ...        .        •.       •        •  .  .  334 

Preparation  of  Samples    .         .        ....        .  .  .  334 

Methods  of  Analysis         .  •      .        .         .        •        •        •  •  •  335 

Interpretation  of  Results .         .         .        .        ...  .  .  343 

References  346 


CHAPTER  XVII 

MILK         .        .        .        ...        .        .        .        .•.'„..        .349 

'Sampling  and  Preservation  of  Samples    .      •  .         .        »                 .  352 

Preliminary  or  Partial  Examination        .        .         .        ,        .        .  353 

Determination  of  Fat,  Proteins,  Milk  Sugar,  and  Ash    .         .         .  356 

Interpretation  of  Results .        '.        .         .  • 362 

Examination  of  Milk  Serum  for  Added  Water        ....  364 

Chemical  Preservatives     .         .         ...         .         .         .         .  365 

References         .         .         .         .         .         .         .         .  366 


CHAPTER  XVIII 

FOOD  PRESERVATIVES    .        .r      .        .        ;. 369 

Formaldehyde  ...........  369 

Hydrogen  Peroxide 372 

Boric  Acid  and  Borates 373 

Fluorides .         .        .         .         .        .  375 

Fluoborates  and  Fluosilicates .  376 

Sulphurous  Acid       .         .        *        .       ' .         .        .        .         ...  377 

Salicylic  Acid    .         .         .         .         ...        .        .         .         .  378 

Benzoic  Acid  and  Benzoates     .         .        .        .        .        .        .        .  385 

Saccharin.         .         . .  388 

Beta-Naphthol .        .     .    .  v      .        >.        .        .        .        .        .         .  389 

Abrastol 389 

Sucrol  or  Dulcin        ........       \  390 

References         .         .        .        ...        .        .        .        .        .  391 


INTRODUCTION 

ULTIMATE  organic  analysis  is  the  determination  of  the  ele- 
ments composing  any  organic  substance.  Proximate  Organic 
analysis  is  the  determination  of  the  compounds  present  in  a 
mixture  or  of  the  radicles  present  in  a  compound. 

Both  ultimate  and  proximate  analysis  are  often  required  in 
the  examination  of  organic  materials.  In  the  case  of  a  complex 
mixture,  however,  proximate  analysis  is  frequently  directed 
to  the  determination  of  the  principal  groups  of  related  com- 
pounds rather  than  of  each  individual  compound  present. 
Thus  in  the  analysis  of  a  food  it  may  sometimes  suffice  to 
determine  the  percentage  of  moisture  and  of  each  of  the  groups 
of  substances  represented  by  the  terms  proteins,  fats,  carbo- 
hydrates, and  ash  constituents. 

In  the  analysis  of  ordinary  animal  and  vegetable  substances 
it  is  usually  difficult  to  make  a  clear  distinction  between  organic 
and  inorganic  constituents,  because  in  many  cases  the  inorganic 
compounds  found  in  the  ash  are  formed  during  combustion, 
the  bases  having  existed  in  combination  with  organic  acids  or 
with  proteins,  while  the  acid  radicles  may  also  have  existed  in 
organic  combination  or  may  have  been  formed  by  the  oxida- 
tion of  the  carbon,  sulphur,  and  phosphorus  of  the  organic 
matter. 

The  chapters  which  follow  are  devoted  chiefly  to  methods 
of  proximate  analysis.  The  determination  of  carbon  and 
hydrogen  by  means  of  the  combustion  furnace  is  doubtless 
already  familiar  to  most  users  of  this  book  and  is  therefore 
omitted,  but  methods  for  determining  nitrogen,  sulphur,  and 
phosphorus  will  be  found  in  Chapter  XIV. 


XVI  INTRODUCTION 

The  sequence  of  topics  is  in  the  main  that  usually  followed 
in  textbooks  of  organic  chemistry,  but  exceptions  are  made 
in  some  cases  in  order  to  bring  together  subjects  which  are 
closely  related  in  practical  interest. 


METHODS  OF 
OKGANIC   ANALYSIS 


METHODS  OF  ORGANIC  ANALYSIS 

CHAPTER   I 
Alcohols 

ALCOHOLS  are  neutral  hydroxyl  derivatives  capable  of  react- 
ing with  acids  to  form  esters,  and  the  most  characteristic 
alcohol  reactions  are  those  involving  the  replacement  of  the 
hydroxyl  by  an  acid  radicle.  For  detailed  discussions  of  the 
analytical  behavior  of  the  hydroxyl  radicle  and  characteristic 
reactions  of  the  different  groups  of  alcohols,  the  reader  is  re- 
ferred to  the  works  of  Meyer,1  and  of  Meyer  and  Tingle,1  while 
Mulliken's  tables1  may  be  followed  in  the  systematic  identifica- 
tion of  individual  pure  preparations. 

Of  the  analytical  methods  involving  reactions  of  the  hydroxyl 
group,  the  more  important  are  those  in  which  the  acetyl  or 
benzoyl  ester  is  formed.  The  preparation  of  the  dinitrobenzo- 
ate  serves  for  the  identification  of  ethyl  alcohol,  and  glycerol 
may  be  determined  quantitatively  by  acetylation.  Often,  how- 
ever, esterification  is  either  not  quantitative  or  is  less  con- 
venient or  less  delicate  than  other  methods  of  determination. 
Thus  the  monatomic  alcohols  containing  less  than  four  carbon 
atoms  mix  freely  with  water  and  are  not  readily  separated  from 
it,  but  may  be  determined  in  aqueous  solution  either  by  physi- 
cal methods  or  by  the  behavior  of  the  alcohols  on  oxidation. 

The  present  chapter  will  deal  with  ethyl  alcohol  and  the  de- 
tection and  determination  of  a  few  of  its  more  important  homo- 
logues.  Glycerol  will  be  considered  in  connection  with  soap  in 
a  later  chapter.  For  more  extended  discussions  of  the  analyti- 
cal chemistry  of  the  alcohols  the  works  cited  at  the  end  of  the 
chapter  may  be  consulted. 

1  The  titles  of  these  and  other  works  of  reference  will  be  found  at  the  end  of 
the  chapter. 

B  1 


£  METHODS    OF   ORGANIC   ANALYSIS 

ETHYL  ALCOHOL 

Pure  ethyl  alcohol  is  a  colorless,  mobile  liquid  of  characteris- 
tic penetrating  odor  and  "  hot "  pungent  taste,  boiling  at  about 
78.4°  and  showing  at  15.56°  (60°  F.),  compared  with  water  at 
the  same  temperature,  a  specific  gravity  of  0.79387  (Bureau  of 
Standards).  It  mixes  with  water  in  all  proportions  and  is  only 
with  great  difficulty  obtained  in  the  anhydrous  or  "  absolute  " 
state.  According  to  Allen  the  presence  of  as  small  proportion 
as  0.5  per  cent  of  water  in  alcohol  is  indicated  by  the  pink 
color  assumed  by  the  liquid  on  introducing  a  crystal  of  potas- 
sium permanganate.  The  so-called  "  absolute  alcohol "  used  in 
analytical  operations  ordinarily  contains  from  0.2  to  1  per  cent 
of  water. 

Alcohol  is  of  great  importance  in  organic  analysis  not  only 
as  a  constituent  to  be  determined  but  also  as  a  solvent  in  ana- 
lytical operations.  It  dissolves  many  organic  substances  which 
are  not  soluble  in  water,  but  inorganic  compounds  insoluble  in 
water  are  usually  also  insoluble  in  alcohol.  As  a  rule  chlorides, 
bromides,  iodides,  and  acetates  are  soluble  in  fairly  strong 
alcohol,  while  carbonates,  borates,  sulphates,  phosphates,  oxa- 
lates,  and  tartrates  are  only  very  sparingly  soluble. 

Three  strengths  of  alcohol  are  recognized  in  the  U.  S. 
Pharmacopoeia  of  1905 : 

"Alcohol"  containing  about  92.3  per  cent  by  weight  or  about 
94.9  per  cent  by  volume  of  actual  ethyl  alcohol  and  about 
7.7  per  cent  by  weight  of  water ;  specific  gravity  about 
0.816  at  60°  F. 

"Absolute  Alcohol"  containing  not  more  than  1  per  cent  by 
weight  of  water;  specific  gravity  not  higher  than  0.798  at 
60°  F. 

"  Diluted  Alcohol "  made  by  mixing  equal  volumes  of  "  alcohol " 

and  water  and  containing  about  41.5  per  cent  by  weight 

or  48.9  per  cent  by  volume  of  actual  ethyl  alcohol ;  specific 

gravity  about  0.936  at  60°  F. 

On  mixing  alcohol  with  water  a  considerable  evolution  of 

heat  takes  place  and  the  volume  of  the  mixture  after  cooling 


ALCOHOLS  6 

is  less  than  the  sum  of  the  volumes  of  the  alcohol  and  water 
mixed.  This  contraction  is  not  uniformly  proportional  to  the 
amount  of  alcohol  in  the  mixture.  Hence  in  mixtures  of  water 
and  alcohol  the  relation  between  the  percentages  of  alcohol  "  by 
volume  "  and  "  by  weight  "  varies  somewhat  with  the  strength 
of  the  solutions.  These  variations  together  with  the  differences 
in  the  density  of  the  supposedly  absolute  alcohol  used  as  stand- 
ard by  the  various  observers  account  for  the  small  discrepancies 
found  on  comparing  the  commonly  used  tables  showing  the  re- 
lation between  density  and  percentage. 

In  the  United  States  the  tables  principally  used  are  (1)  those  of 
the  U.  S.  Bureau  of  Standards  recalculated  from  the  determina- 
tions made  by  Mendelejeff  and  (2)  those  based  on  Squibb's  de- 
terminations, which  have  been  very  generally  used  in  this  country 
and  incorporated  in  the  methods  of  the  Association  of  Official 
Agricultural  Chemists  and  the  U.  S.  Pharmacopoeia. 

The  official  chemists  have  under  consideration  the  question 
of  substituting  the  tables  of  the  Bureau  of  Standards  for  those 
now  official.  The  two  systems  differ  chiefly  in  the  conditions 
taken  as  standard,  the  final  results  when  properly  calculated 
being  very  nearly  the  same,  especially  for  solutions  containing 
less  than  25  per  cent  of  alcohol.  The  methods  and  data  given 
in  this  chapter  provide  for  the  use  of  either  system,  Table  I 
(beyond)  being  from  the  Bureau  of  Standards,  and  Table  II 
condensed  from  the  tables  of  the  Official  Agricultural  Chemists 
and  of  the  U.  S.  Pharmacopoeia. 

The  table  of  Morley  (J".  Am.  Ohem.  Soc.,  26, 1185-1193),  also 
based  on  the  observations  of  Mendelejeff,  gives  the  specific 
gravity  of  alcohol  for  each  integral  percentage  by  weight  and 
for  each  degree  of  the  hydrogen  thermometer  from  15°  to  22°  C. 

For  revenue  purposes,  both  in  the  United  States  and  Great 
Britain,  the  strength  of  alcoholic  liquors  is  expressed  in  terms 
of  "  proof  spirit,"  but  the  term  has  different  meanings  in  the  two 
countries. 

American  proof  spirit  is  defined  by  Section  3249  of  the  Re- 
vised Statutes  of  the  United  States  as  follows:  "  Proof  spirit 
shall  be  held  to  be  that  alcoholic  liquor  which  contains  one  half 


4  METHODS    OF   ORGANIC    ANALYSIS 

its  volume  of  alcohol  of  a  specific  gravity  of  seven  thousand 
nine  hundred  and  thirty-nine  ten-thousandths  at  60°  F."  It  is 
therefore  practically  50  per  cent  by  volume  or  about  42.5  per  cent 
by  weight. 

British  proof  spirit  is  denned  by  Parliament  as  having  such  a 
density  that  at  57°  F.  thirteen  volumes  shall  weigh  the  same  as 
twelve  volumes  of  water  at  the  same  temperature.  This  cor- 
responds to  about  49.2  per  cent  by  weight. 

"Rectified  spirit"  of  the  British  Pharmacopoeia  has  84  per 
cent  by  weight  and  British  "methylated  spirit"  consists  of  nine 
parts  of  rectified  spirit  to  one  part  of  commercial  "  wood  spirit  " 
or  "  wood  naphtha,"  the  latter  containing,  according  to  the 
observations  of  Thorpe  and  Holmes,  from  72  to  80  per  cent  of 
methyl  alcohol  by  volume. 

Denatured  Alcohol 

A  statute  of  the  United  States  of  June  7,  1906,  provides  that 
domestic  alcohol  may  be  withdrawn  from  bond  for 'use  in  the 
arts  and  industries,  and  for  fuel,  light,  and  power,  without  the 
payment  of  an  internal  revenue  tax,  on  condition  that  it  shall 
have  been  denatured  by  the  admixture  of  some  material  which 
destroys  its  character  as  a  beverage  and  renders  it  unfit  for 
liquid  medicinal  purposes. 

The  regular  formula  for  denaturing  alcohol  is  as  follows  : 
To  100  parts  by  volume  of  ethyl  alcohol  (not  less  than  90  per 
cent  strength)  add  10  parts  of  approved  methyl  (wood)  alcohol 
and  0.5  part  of  approved  benzine.  Such  alcohol  is  classed  as 
completely  denatured  and  becomes  a  regular  article  of  commerce. 
The  wood  alcohol  used  in  denaturing  must  have  a  specific 
gravity  not  above  0.830  at  60°  F.  (corresponding,  if  impurities 
be  neglected,  to  about  88  per  cent  strength,  or  to  91°  on  Tralles 
scale)  and  meet  several  other  requirements  which  include  maxi- 
mum and  minimum  limits  for  acetone  and  bromine-absorbing 
constituents  and  a  maximum  limit  for  esters. 

The  Bureau  of  Internal  Revenue  of  the  Treasury  Department 
establishes  the  detailed  regulations  governing  both  the  regular 
method  of  denaturing  and  the  special  formulas  permitted  in  the 


ALCOHOLS  5 

several  classes  of  industries  in  which  tax-free  alcohol  may  be 
used,  but  for  which  the  alcohol  denatured  by  the  regular  formula 
would  not  be  suitable.  These  formulae  and  specifications  are 
given  in  Regulation  No.  30  of  the  U.  S.  Internal  Revenue, 
together  with  the  supplementary  regulations  and  the  series  of 
"Treasury  Decisions." 

DETECTION  AND  IDENTIFICATION  OF  ETHYL  ALCOHOL 

Lieberis  Iodoform   Test 

The  "  iodoform  test,"  while  not  distinctive,  is  often  useful. 
It  may  be  carried  out  as  follows  :  To  10  cc.  of  the  clear  liquid 
to  be  tested,  add  5  or  6  drops  of  10  per  cent  solution  of  sodium 
or  potassium  hydroxide,  heat  to  about  50°  C.,  and  add  drop  by 
drop  with  constant  shaking  a  saturated  solution  of  iodine  in 
aqueous  potassium  iodide  until  the  liquid  becomes  just  per- 
manently yellowish  brown,  then  carefully  decolorize  by  adding 
more  of  the  hydroxide  solution,  avoiding  excess.  If  alcohol 
were  present,  iodoform  gradually  separates  out  as  a  yellow  or 
yellowish  white  crystalline  deposit.  Even  when  very  little 
iodoform  is  produced  its  odor  can  usually  be  recognized. 
While  the  test  is  quite  delicate,  the  appearance  of  a  precipitate 
of  iodoform  does  not  prove  the  presence  of  ethyl  alcohol,  since 
it  may  result  from  various  other  compounds,  especially  acetone, 
aldehydes,  and  the  propyl  and  butyl  alcohols.  If  the  original 
liquid  contained  carbohydrate  or  organic  acid,  it  should  be 
neutralized,  distilled,  and  the  first  portion  of  the  distillate  used 
for  the  test. 

Ethyl  Dinitrobenzoate  Test 

As  a  specific  test  for  ethyl  alcohol  Mulliken1  recommends 
the  preparation  of  Ethyl  3,  5-Dinitrobenzoate  as  follows  : 

Heat  together  gently  in  a  three-inch  test  tube  held  over  a 
small  flame,  0.15  gm.  of  3,  5-dinitrobenzoic  acid  and  0.20  gm. 
of  phosphorus  pentachloride.  When  signs  of  chemical  action 
are  seen,  remove  the  heat  for  a  few  seconds.  Then  heat  again, 

1  Identification  of  Pure  Organic  Compounds,  Vol.  I,  p.  168. 


D  METHODS  OF  ORGANIC  ANALYSIS 

boiling  the  liquefied  mixture  very  gently  for  one  minute.  Pour 
out  on  a  very  small  watchglass  and  allow  to  solidify.  As  soon 
as  solidification  occurs,  remove  the  liquid  phosphorus  oxy- 
chloride,  with  which  the  crystalline  mass  is  impregnated,  by 
rubbing  the  latter  between  two  small  pieces  of  porous  tile. 
Place  the  powder  in  a  dry  five-  or  six-inch  test  tube.  Allow 
four  drops  of  the  alcohol  (which  must  contain  not  more  than 
about  10  per  cent  of  water)  to  fall  upon  it,  and  then  stopper 
the  tube  tightly  without  delay.  Immerse  the  lower  part  of  the 
test  tube  in  water  having  a  temperature  of  75°-85°.  Shake 
gently,  and  continue  the  heating  for  ten  minutes. 

To  purify  the  ester  produced  in  the  reaction,  crush  with  a 
stirring  rod  any  hard  lumps  which  may  form  when  the  mixture 
cools  and  boil  gently  with  15  cc.  of  methyl  alcohol  (2  :  1)  until 
all  is  dissolved,  or  for  a  minute  or  two.  Filter  boiling  hot  if 
the  solution  is  not  clear.  Cool,  shake  and  filter.  Wash  with 
3  cc.  cold  methyl  alcohol  (2:1).  Recrystallize  from  9  cc.  of 
boiling  methyl  alcohol  (2:1).  Wash  with  2  cc.  of  the  same 
solvent.  Spread  out  the  product  on  a  piece  of  tile.  Allow  to 
become  air-dry,  and  determine  the  melting  point. 

Ethyl  3,  5-Dinitrobenzoate,  the  product  of  this  test,  crystal- 
lizes in  white  needles  melting  at  92°-93°  (uncorr.). 

The  corresponding  derivatives  of  methyl,  propyl,  butyl,  and 
isobutyl  alcohols  melt  at  107.5°  (uncorr.),  73°-73.5°  (uncorr.), 
64°  (uncorr.),  and  83°-83.5°  (uncorr.),  respectively. 

Other  Methods 

The  methods  recommended  by  Bacon  as  being  especially 
adapted  to  the  detection  and  determination  of  such  small 
amounts  of  alcohol  as  may  be  present  as  the  result  of  incipient 
spoilage  in  food  material  are  described  later  in  this  chapter. 

DETERMINATION  OF  ETHYL  ALCOHOL 

As  already  stated,  the  complete  separation  of  ethyl  alcohol 
from  water  is  very  difficult.  The  quantitative  determination 
of  the  alcohol  is  therefore  carried  out  in  water  solution  and  is 
usually  accomplished  by  one  of  the  following  methods:  from 


ALCOHOLS  7 

(1)  the  density,  (2)  the  index  of  refraction,  (3)  the  boiling 
point  of  the  solution,  or  (4)  by  quantitative  oxidation  of  the 
alcohol  to  acetic  acid  by  heating  in  acid  solution  with  potassium 
dichromate. 

The  first  and  second  methods  are  seriously  influenced  by  the 
presence  of  any  substance  other  than  the  alcohol  and  water  and 
are  ordinarily  applied  only  after  distillation  ;  the  third  method 
is  less  influenced  by  dissolved  solids,  and  for  approximate  work 
may  be  applied  directly  to  ordinary  alcoholic  liquors,  but  is 
not  so  accurate  as  the  specific  gravity  method;  the  fourth 
method  is  applicable  only  in  the  absence  of  any  other  substance 
capable  of  reacting  with  the  acid-dichromate  mixture,  and  is 
not  readily  adaptable  to  the  determination  of  more  than  small 
amounts. 

Generally  the  most  accurate  and  satisfactory  method  is  to 
separate  the  alcohol  from  substances  other  than  water  by  dis- 
tillation and  find  the  amount  of  alcohol  in  the  distillate  by  a 
careful  determination  of  its  specific  gravity. 

Preparations  containing  chloroform,  ether,  or  essential  oils 
may  be  treated  as  follows : 1  Dilute  25  cc.  of  the  sample  with 
water  to  about  100  cc.  in  a  separatory  funnel,  add  sodium 
chloride  to  saturation  and  then  50  to  80  cc.  of  light  petroleum 
distillate  (boiling  below  60°).  Shake  vigorously  for  five  min- 
utes, allow  to  stand  for  half  an  hour,  draw  off  the  lower  layer 
into  another  separatory  funnel,  wash  again  in  the  same  way 
with  a  small  amount  of  petroleum  ether,  and  then  draw  off  into 
a  distillation  flask.  Unite  the  petroleum  ether  layers,  wash 
with  two  successive  portions  each  25  cc.  of  water  saturated 
with  salt,  adding  these  washings  to  the  main  solution.  The 
alcohol  remains  dissolved  in  the  aqueous  salt  solution,  which 
(after  neutralizing  if  necessary)  may  be  distilled  as  described 
below. 

Carbon  dioxide  if  present  in  large  amount  should  be  removed 
as  well  as  possible  in  advance  by  shaking  the  liquor  in  a  large 
flask  at  room  temperature  and  avoiding  the  froth  when  taking 
the  portions  for  analysis. 

. l  Thorpe  and  Holmes :  J.  Chem.  8oc.,  1903,  83,  314. 


8  METHODS  OF  ORGANIC  ANALYSIS 

Unless  the  appearance  or  odor  of  the  sample  suggests  the 
presence  of  carbon  dioxide  or  of  chloroform,  ether,  or  essential 
oil,  it  is  usually  permissible  to  proceed  with  the  determination 
as  described  below  without  subjecting  the  sample  to  any  of  the 
above  preliminary  treatment. 

Specific  Gravity  Method 

In  the  case  of  a  fermented  liquor  or  other  sample  containing 
less  than  25  per  cent  of  alcohol  (Note  1),  dry  and  weigh  a  100 
cc.  graduated  flask,  fill  to  the  mark  with  the  sample  and  weigh 
(Note  2);  transfer  to  a  distilling  flask  of  about  300  cc.  ca- 
pacity, rinsing  the  measuring  flask  with  about  50  cc.  of  water, 
bringing  the  total  volume  in  the  distilling  flask  to  about  150 
cc.  Drop  into  the  solution  a  small  piece  of  delicate  litmus 
paper,  or  better  a  few  milligrams  of  solid  phenolphthalein,  and 
neutralize  with  a  dilute  solution  of  sodium  or  potassium  hy- 
droxide in  order  that  no  volatile  acid  may  be  distilled  with  the 
alcohol  (Note  3).  Connect  the  distilling  flask  with  a  well- 
cooled  condenser  and  distill,  collecting  the  distillate  in  a  100  cc. 
flask,  preferably  the  one  used  in  measuring  the  sample.  The 
distillate  should  not  be  unnecessarily  exposed  to  the  air  but 
should  be  conducted  well  into  the  receiving  flask.  If  the  con- 
denser is  vertical  or  nearly  so,  it  is  only  necessary  to  support 
the  flask  in  such  a  position  that  the  condenser  tube  projects 
into  the  neck  of  the  flask,  otherwise  an  adapter  may  be  used 
(Note  4).  The  apparatus  may  be  arranged  as  shown  in 
Fig.  1. 

When  the  distillate  amounts  to  nearly  100  cc.  remove  the 
receiver ;  and  if  the  alcohol  is  to  be  reported  in  percentage  by 
volume,  the  temperature  should  be  brought  to  that  at  which  the 
sample  was  measured  out  for  analysis  and  water  then  added  to 
bring  the  distillate  exactly  to  100  cc.,  but  if  the  results  are  to 
be  reported  by  weight,  this  adjustment  is  not  important.  See 
that  the  flask  is  clean  and  dry  outside,  weigh  and  subtract  the 
weight  of  the  empty  flask  as  found  at  the  beginning  of  the 
experiment.  Mix  the  distillate  well  by  shaking,  and  by  means 
of  a  pyknometer  (Note  5)  determine  carefully  its  specific 


ALCOHOLS  9 

gravity  to  the  fifth  decimal  place,  with  special  attention  to  the 
accurate  control  of  the  temperature,  which  should  be  governed 
according  to  the  table  which  is  to  be  used  in  finding  the  per- 
centage of  alcohol  from  the  specific  gravity  (Note  6).  Find 
in  Table  1  the  percentage  of  alcohol  by  weight,  or  in  Table  2 


•      FIG.  1.  —  Arrangement  of  distillation  apparatus  for  alcohol  determination. 

the  percentages  by  weight  and  by  volume,  corresponding  to 
this  specific  gravity. 

The  percentage  by  weight  of  alcohol  in  the  distillate,  multi- 
plied by  the  weight  of  the  latter,  shows  the  actual  weight  of 
alcohol  distilled  over,  and  this  divided  by  the  weight  of  sample 
taken  gives  the  percentage  by  weight  of  alcohol  in  the  liquor. 
The  percentage  by  volume  is  of  course  the  same  for  the  origi- 
nal sample  as  for  the  distillate,  if  both  are  measured  in  the 
same  flask  at  the  same  temperature. 

Note  1. — If  the  sample  contains  more  than  25  per  cent  of 
alcohol,  a  proportionately  smaller  amount  than  100  cc.  should 
be  taken  for  the  alcohol  determination.  Of  cordials,  50  cc., 
and  of  distilled  liquors  or  commercial  alcohol,  25  cc.,  will  usu- 
ally be  a  suitable  amount.  If  results  are  to  be  expressed  in 


10  METHODS   OF   ORGANIC   ANALYSIS 

percentage  by  weight,  the  weight  as  well  as  the  volume  of  the 
sample  taken  must  of  course  be  known. 

Note  2.  —  If  the  specific  gravity  of  the  sample,  is  known,  or 
if  the  results  of  the  analysis  are  to  be  expressed  only  in  per- 
centage by  volume  or  grams  per  100  cc.,  the  portion  desired 
for  the  determination  may  be  measured  by  means  of  a  pipette. 
If,  however,  the  same  100  cc.  flask  is  used  for  measuring  the 
sample  and  for  the  distillate  and  each  is  weighed  as  well  as 
measured,  the  accuracy  of  the  result  whether  expressed  in  per- 
centage by  weight  or  by  volume  will  be  independent  of  the 
accuracy  of  calibration  of  this  measuring  flask. 

Note  3.  —  Most  alcoholic  beverages,  unless  they  have  under- 
gone acetic  fermentation,  contain  too  little  volatile  acid  to 
affect  materially  the  result  of  the  alcohol  determination.  It  is, 
however,  not  safe  to  neglect  the  neutralization  unless  volatile 
acids  are  known  to  be  absent. 

Note  4.  —  A  suitable  arrangement  for  this  distillation  is 
shown  in  Fig.  1.  The  bulb  in  the  connecting  tube  serves  to 
prevent  the  mechanical  carrying  over  of  spray,  and  permits 
the  distillation  to  be  carried  on  at  a  fairly  rapid  rate,  provided 
a  sufficient  stream  of  cold  water  flows  through  the  condenser 
to  thoroughly  chill  the  distillate.  If  a  distilling  flask  having 
side  tube  on  the  neck  is  used,  the  distillation  should  be  con- 
ducted more  slowly,  in  order  to  avoid  danger  from  spray,  and  if 
possible  a  flask  should  be  selected  which  has  the  exit  tube  high 
on  the  neck.  If  it  is  desired  to  expel  the  alcohol  quickly,  the 
solution  may  be  saturated  with  salt  before  distilling. 

Note  5.  —  On  account  of  the  importance  of  exact  control  of 
temperature,  it  is  desirable  to  use  either  a  thin-walled  Sprengel- 
Ostwald  pyknometer,  which  may  be  hung  in  water  of  the  de- 
sired temperature  for  adjustment,  or  a  pyknometer  bottle  of 
the  type  carrying  a  thermometer  in  the  ground  glass  stopper 
and  having  a  capillary  side  tube  for  overflow  and  adjustment. 
In  either  case  the  pyknometer  is  calibrated  by  weighing  first 
empty  and  dry,  then  filled  with  recently  boiled  distilled  water 
of  known  temperature.  When  using  the  pyknometer,  care  must 
be  exercised  to  avoid  warming  it  by  contact  with  the  hand. 


ALCOHOLS 


11 


\ 


FIG.  2.  —  The  Ostwald 
pyknometer. 


The  Ostwald  pyknometer  (Fig.  2)  has  one  arm  (^4.)  of  uni- 
form bore,  while  the  other  arm  carries  a  pipette  body  (B)  and 
ends  in  a  capillary  tip  ((7).  It  is  ^ 

filled  by  attaching  a  small  rubber 
tube  at  0  and  drawing  the  liquid 
through  A  until  the  entire  pyknome- 
ter is  full,  when  it  is  suspended  in 
water  at  the  desired  temperature, 
and  when  this  is  reached  the  adjust- 
ment is  made  by  touching  the  point 
<7  with  filter  paper  until  just  enough 
liquid  is  removed  to  bring  the  menis- 
cus in  A  to  the  graduation  mark  M ; 
then  cap  the  tips  (if 
caps  are  provided), 
remove  and  care- 
fully wipe  the  pyk- 
nometer, suspend  it 
from  the  hook  over 
the  balance  pan,  and  weigh. 

The  specific  gravity  bottle  of  the  form  shown 
in  Fig.  3,  while  not  so  good  in  principle  as  the 
Ostwald  pyknometer,  is  more  widely  used  in 
analytical  laboratories.     The  thermometer  of 
such  a  bottle  should  (unless  it  bears  a  "con- 
jf       trol  stamp  ")  be  tested,  by  comparison  with  a 
JL          thermometer  known  to  be  accurate,  at  the 
>>     temperature    at   which    it    is   to   be    used. 
This   pyknometer   should  always  be  filled 
either  at  or  slightly  below  the  temperature 
at  which  the  weighing  is  to  be  made.     Fill 
to  about  the  middle  of  the  ground  portion 
of  the  neck  so  that  when  the  stopper  is  in- 
serted  the   liquid   will   overflow  not   only 
around  the  stopper,  but  also  through  the 
side  tube.     The  stopper  should  be  inserted 
snugly  but  without  unnecessary  force.     As 


FIG.  3.  —  Specific  grav- 
ity bottle  with  ther- 
mometer in  stopper. 


12  METHODS  OF  ORGANIC  ANALYSIS 

soon  as  the  stopper  is  in  place,  wipe  off  the  tip  of  the  side  tube, 
place  the  pyknometer  in  water  (or  air)  maintained  at  the 
desired  temperature,  and  notice  whether  any  further  overflow 
occurs.  When  the  reading  of  the  thermometer  in  the  pyknom- 
eter and  the  position  of  the  surface  of  the  liquid  at  the  tip  of 
the  side  tube  remain  constant,  the  contents  are  evidently  at  the 
same  temperature  with  the  surrounding  water  or  air.  The 
glass  cap  is  now  placed  over  the  tip,  the  pyknometer  carefully 
wiped  with  a  clean,  dry  cloth,  weighed,  and  the  weight  of  the 
empty  pyknometer  deducted. 

Note  6.  — The  pyknometer,  having  been  weighed  with  water 
as  just  described,  is  either  dried  or  repeatedly  rinsed  with  small 
portions  of  the  distillate  and  then  filled  with  the  latter  at  a 
little  below  the  standard  temperature  for  the  alcohol  table  which 
is  to  be  used,  then  allowed  to  warm  slowly  up  to  the  exact  tem- 
perature desired  and  at  exactly  this  temperature  the  volume 
is  carefully  adjusted,  the  pyknometer  capped,  wiped,  and 
weighed  as  described  in  Note  5. 

To  use  Table  1,  correct  the  weighings  of  water  and  of  distillate 
by  adding  0.00106  gram  to  each  gram  of  water  or  distillate 
which  the  pyknometer  apparently  contains.  (This  corrects  for 
the  buoyant  effect  of  the  air  when  weighings  are  made  with  brass 
weights  in  air  of  average  humidity  at  room  temperature.) 
To  simplify  calculations  adopt  either  20°  or  25°  as  the  tempera- 
ture for  adjusting  the  pyknometer  both  with  water  and  with 
the  distillate.  Divide  the  corrected  weight  of  distillate  by  the 
corrected  weight  of  water,  thus  obtaining  the  specific  gravity 
at  f -j}  or  |f.  Divide  this  by  the  density  of  water  at  20° 
(0.998234)  or  25°  (0.997077)  as  the  case  may  be  to  obtain  the 
density  of  the  distillate  at  -^  or  %£-,  from  which  by  interpola- 
tion in  Table  1  find  the  percentage  by  weight  of  alcohol  in 
the  distillate  to  the  second  decimal  place. 

If  for  any  reason  the  temperature  of  adjustment  has  been  be- 
tween 20°  and  25°,  the  density  of  water  at  the  known  temperature 
of  adjustment  can  be  found  from  Table  3  below  and  the  cor- 
rected weight  of  water  calculated  thus  to  4°  can  be  used  in  find- 
ing the  density  of  distillate  for  the  temperature  of  adjustment. 


ALCOHOLS 


13 


TABLE  1.  —  DENSITY  OF  MIXTURES  OF  ETHYL  ALCOHOL  AND  WATER 
(BUREAU  OF  STANDARDS) 


Per  cent 
alcohol 
by 

weight 

D^i 

D¥ 

D2jf- 

Per  cent 
alcohol 

t>y 

weight 

&f 

&¥ 

B* 

0 

0.99913 

0.99824 

0.99708 

50 

0.917S7 

0.91386 

0.90983 

1 

.99725 

.99636 

.99521 

51 

.91566 

.91164 

.90758 

2 

.99543 

.99453 

.99338 

52 

.91344 

.90940 

.90533 

3 

.99366 

.99274 

.99159 

53 

.91120 

.90715 

.90307 

4 

.99197 

.99102 

.98984 

54 

.90895 

.90488 

.90079 

5 

.99033 

.98936 

.98815 

55 

.90670 

.90262 

.89851 

6 

.98877 

.98776 

-  .98651 

56 

.90443 

.90034 

.89622 

1 

.98726 

.98620 

.98491 

57 

.90215 

.89805 

.89392 

8 

.98581 

.98470 

.98336 

58 

.89987 

.89576 

.89162 

9 

.98442 

.98325 

.98185 

59 

.89758 

.89346 

.88931 

10 

.98307 

.98185 

.98038 

60 

.89528 

.89115 

.88700 

11 

.98176 

.98047 

.97893 

61 

.89297 

.88883 

.88467 

12 

.98049 

.97913 

.97752 

62 

.89066 

.88651 

.88234 

13 

.97925 

.97781 

.97612 

63 

.88834 

.88418 

.88000 

14 

.97803 

.97651 

.97474 

64 

.88601 

.88185 

.87766 

15 

.97683 

.97522 

.97336 

65 

.88368 

.87950 

.87530 

16 

.97563 

.97393 

.97199 

66 

.88134 

.87716 

.87295 

17 

.97444 

.97264 

.97061 

67 

.87899 

.87480 

.87058 

18 

.97324 

.97134 

.96922 

68 

.87664 

.87244 

.86821 

19 

.97203 

.97003 

.96782 

69 

.87428 

.87008 

.86583 

20 

.97080 

.96870 

.96640 

70 

.87192 

.86770 

.86344 

21 

.96956 

.96736 

.96497 

71 

.86954 

.86532 

.86105 

22 

.96829 

.96599 

.96352 

72 

.86716 

.86292 

.85864 

23 

.96699 

.96459 

.96203 

73 

.86477 

.86052 

.85622 

24 

.96566 

.96317 

.96052 

74 

.86237 

.85812 

.85380 

25 

.96430 

.96171 

.95897 

75 

.85997 

.85570 

.85137 

26 

.96289 

.96021 

.95739 

.  76 

.85755 

.85328 

.84893 

2T 

.96145 

.95868 

.95577 

77 

.85513 

.85084 

.84648 

28 

.95997 

.95711 

.95412 

78 

.85270 

.84840 

.84403 

29 

.95845 

.95550 

.95244 

79 

.85026 

.84595 

.84157 

30 

.95688 

.95385 

.95071 

80 

.84781 

.84349 

.83909 

31 

.95526 

.95215 

.94894 

81 

.84534 

.84101 

.88660 

82 

k  .95360 

.95042 

.94713 

82 

.84286 

.83852 

.83410 

88 

.95191 

.94865 

.94529 

83 

.84037 

.83602 

.83159 

34 

.95017 

.94684 

.94342 

84 

.83786 

.83350 

.82906 

35 

.94839 

.94499 

.94152 

85 

.83534 

.83097 

.82652 

36 

.94657 

.94311 

.93957 

86 

.83279 

.82842 

.82396 

37 

.94471 

.94119 

.93760 

87 

.83022 

.82583 

.82137 

38 

.94282 

.93924 

.93560 

88 

.82762 

.82323 

.81876 

39 

.94089 

.93725 

.93356 

89 

.82500 

.82060 

.81613 

40 

.93893 

.93524 

.93151 

90 

.82235 

.81795 

.81348 

41 

.93694 

.93320 

.92943 

91 

.81966 

.81527 

.81080 

42 

.93491 

.93113 

.92732 

92 

.81694 

.81255 

.80809 

43 

.93286 

.92904 

.92519 

93 

.81418 

.80979 

.80534 

44 

,93078 

.92693 

.92305 

94 

.81138 

.80700 

.80256 

45 

.92868 

.92480 

.92088 

95 

.80854 

.80417 

.79974 

46 

.92655 

.92264 

.91870 

96 

.80564 

.80129 

.79689 

47 

.92441 

.92047 

.91650 

97 

.80271 

.79838 

.79400 

48 

.92225 

.91828 

.91429 

98 

.79972 

.79541 

.79106 

49 

.92006 

.91608 

.91207 

99 

.79668 

.79240 

.78809 

50 

.91787 

.91386 

.90983 

100 

.79358 

.78933 

.78507 

=  Density  at  15°  C.  referred  to  water  at  4°  C. 


14  METHODS   OF   ORGANIC   ANALYSIS 

Suppose  this  is  found  to  be  0.98123  at  -^-;  by  comparing  the 
figures  in  Table  1  for  ^°-  and  %£-  at  about  this  density  it  will  be 
seen  that  the  decrease  of  density  in  alcohol  of  this  strength  is 
about  0.000294  for  each  1°  C.  between  20°  and  25°.  Hence  by 
adding  3  x  0.000294  to  0.98123  (the  density  at  -\3-)  we  obtain 
0.98211  as  the  density  at  -^j0-,  which  is  found  from  Table  1  to 
correspond  to  9.81  per  cent  of  alcohol  by  weight.  The  interpo- 
lation for  temperature  is  apt  to  introduce  a  slight  error,  for 
which  reason  as  well  as  to  simplify  calculation  it  is  preferable 
to  adjust  the  pyknometer  always  at  20°  or  25°  if  practicable. 

To  use  Table  2  find  the  weight  of  distillate  which  the  pyk- 
nometer holds  at  15.56°  and  divide  by  the  weight  of  water 
which  it  holds  at  the  same  temperature.  In  this  case  the 
weighings  are  not  corrected  for  displaced  air  (since  this  correc- 
tion was  apparently  not  made  in  determining  the  data  from 
which  the  table  was  constructed),  but  it  is  necessary  to  exer- 
cise great  care  in  the  control  of  the  temperature,  since  15.56° 
is  much  below  the  usual  working  temperature  of  the  laboratory. 
The  water  content  of  the  pyknometer  may  be  observed  at  a 
higher  temperature  and  calculated  to  15.56°  from  the  data  in 
Table  3 ;  but  since  with  rising  temperature  alcohol  expands  at 
an  uneven  rate  the  adjustment  of  the  pyknometer  with  the  dis- 
tillate must  be  at  or  near  the  standard  temperature,  though  if 
only  a  few  degrees  from  standard  the  temperature  may  be 
taken,  the  weight  divided  by  the  weight  of  water  at  15.56°  to 
find  the  "  apparent  specific  gravity,"  then  correct  this  by  the 
following  empirical  formula  given  by  Allen,1  in  which  D  is  the 
specific  gravity  at  15.56°,  Df  the  "apparent  specific  gravity," 
and  d  the  difference  (in  degrees  centigrade)  between  15.56°  and 
the  temperature  at  which  Df  was  determined : 

D  =  D'  ±d  (o.  00014  + 


The  correction  is  added  to  or  subtracted  from  Dr  according 
as  the  pyknometer  was  filled  and  adjusted  at  a  temperature 
above  or  below  the  standard. 

1  Commercial  Organic  Analysis,  Vol.  I  (3d  Ed.)  p.  93. 


ALCOHOLS 


15 


TABLE  2.  —  PERCENTAGE  OF  ALCOHOL  BY  WEIGHT  AND  BY  VOLUME 

[Recalculated  from  the  determinations  of  Gilpin,  Drinkwater,  and  Squibb,  by 
EDGAR  RICHARDS.] 


Specific  gravity 
at|8°'F. 

Per  cent  alcohol 
by  volume 

Per  cent  alcohol 
by  weight 

Specific  gravity 
at  eg0  F. 

Per  cent  alcohol 
by  volume 

Per  cent  alcohol 
by  weight 

Specific  gravity 
atf8°F. 

Per  cent  alcohol 
by  volume 

Per  cent  alcohol 
by  weight  . 

1.00000 

0.00 

0.00 

0.99281 

5.00 

4.00 

0.98660 

10.00 

8.04 

0.99984 

.10 

.08 

268 

.10 

.08 

649 

.10 

.12 

968 

.20 

.16 

255 

.20 

.16 

637 

.20 

.20 

953 

.30 

.24 

241 

.30 

.24 

626 

.30 

.29 

937 

.40 

.32 

228 

.40 

.32 

614 

.40 

.37 

.99923 

0.50 

0.40 

.99215 

5.50 

4.40 

.98603 

10.50 

8.45 

907 

.60 

.48 

202 

.60 

.48 

592 

.60 

.53 

892 

.70 

.56 

'189 

.70 

.56 

580 

.70 

.61 

877 

.80 

.64 

175 

.80 

.64 

569 

.80 

.70 

861 

.90 

.71 

162 

.90 

.72 

557 

.90 

.78 

.99849 

1.00 

0.79 

.99149 

6.00 

4.80 

.98546 

11.00 

8.86 

834 

.10 

.87 

136 

.10 

.88 

535 

.10 

.94 

819 

.20 

.95 

123 

.20 

.96 

524 

.20 

9.02 

805 

.30 

1.03 

111 

.30 

5.05 

513 

.30 

.11 

790 

.40 

.11 

098 

.40 

.13 

502 

.40 

.19 

.99775 

1.50 

1.19 

.99085 

6.50 

5.21 

.98491 

11.50 

9.27 

760 

.60 

.27 

072 

.60 

.29 

479 

.60 

.35 

745 

.70 

.35 

059 

.70 

.37 

468 

.70 

.43 

731 

.80 

.43 

047 

.80 

.45 

457 

.80 

.51 

716 

.90 

.51 

034 

.90 

.53 

446 

.90 

.59 

.99701 

2.00 

1.59 

.99021 

7.00 

5.61 

.98435 

12.00 

9.67 

687 

.10 

.67 

009 

.10 

.69 

424 

.10 

.75 

672 

.20 

.75 

.98996 

.20 

.77 

413 

.20 

.83 

658 

.30 

.83 

984 

.30 

.86 

402 

.30 

.92 

643 

.40 

.91 

971 

.40 

.94 

391 

.40 

10.00 

.99629 

2.50 

1.99 

.98959 

7.50 

6.02 

.98381 

12.50 

10.08 

615 

.60- 

2.07 

947 

.60 

.10 

370 

.60 

.16 

600 

.70 

.15 

934 

.70 

.18 

359 

.70 

.24 

586 

.80 

.23 

922 

.80 

.26 

348 

.80 

.33 

571 

.90 

.31 

909 

.90 

.34 

337 

.90 

.41 

.99557 

3.00 

2.39 

.98897 

8.00 

6.42 

.98326. 

13.00 

10.49 

543 

.10 

.47 

885 

.10 

.50 

315 

.10 

.57 

529 

.20 

.55 

873 

.20 

.58 

305 

.20 

.65 

515 

.30 

.64 

861 

.30 

.67 

294 

.30 

.74 

501 

.40 

.72 

849 

.40 

.75 

283 

.40 

.82 

.99487 

3.50 

2.80 

.98837 

8.50 

6.83 

.98273 

13.50 

10.90 

473 

.60 

.88 

825 

.60 

.91 

262 

.60 

.98 

459 

.70 

.96 

813 

.70 

.99 

251 

.70 

11.06 

445 

.80 

3.04 

801 

.80 

7.07 

240 

.80 

.15 

431 

.90 

.12 

789 

.90 

.15 

230 

.90 

.23 

.99417 

4.00 

3.20 

.98777 

9.00 

7.23 

.98219 

14.00 

11.31 

403 

.10 

.28 

765 

.10 

.31 

209 

.10 

.39 

390 

.20 

.36 

754 

20 

.39 

198 

.20 

.47 

376 

.30 

.44 

742 

.30 

.48 

188 

.30 

.56 

363 

.40 

.52 

730 

.40 

.56 

177 

.40 

.64 

.99349 

4.50 

3.60 

.98719 

9.50 

7.64 

.98167 

14.50 

11.72 

335 

.60 

.68 

707 

.60 

.72 

156 

.60 

.80 

322 

.70 

.76 

695 

.70 

.80 

146 

.70 

.88 

308 

.80 

.84 

683 

.80 

.88 

135 

.80 

.97 

295 

.90 

.92 

672 

.90 

.96 

125 

.90 

12.05 

16 


METHODS   OF   ORGANIC    ANALYSIS 


PERCENTAGE  OF  ALCOHOL  BY  WEIGHT  AND  BY  VOLUME.  —  Continued. 

[Recalculated  from  the  determinations  of  Gilpin,  Drinkwater,  and  Squibb,  by 
EDGAR  RICHARDS.] 


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12.13 

0.97608 

20.00 

16.26 

0.97097 

25.00 

20.43 

104 

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598 

.10 

.34 

086 

.10 

.51 

093 

.20 

.29 

588 

.20 

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568 

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.20 

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969 

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980 

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477 

.30 

.34 

959 

.30 

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970 

.40 

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467 

.40 

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949 

.40 

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.97960 

16.50 

13.37 

.97457 

21.50 

17.51 

.96937 

26.50 

21.69 

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.60 

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446 

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940 

.70 

.53 

436 

.70 

.67 

915 

.70 

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929 

.80 

.62 

426 

.80 

.76 

905 

.80 

.94 

919 

.90 

.70 

416 

.90 

.84 

894 

.90 

22.03 

.97909 

17.00 

13.78 

.97406 

22.00 

17.92 

.96883 

27.00 

22.11 

899 

.10 

.86 

396 

.10 

18.00 

872 

.10 

.20 

889 

.20 

.94 

386 

.20 

.09 

861 

.20 

.28 

879 

.30 

14.03 

375 

.30 

.17 

850 

.30 

.37 

869 

.40 

.11 

365 

.40 

.26 

839 

.40 

.45 

.97859 

17.50 

14.19 

.97355 

22.50 

18.34 

.96828 

27.50 

22.54 

848 

.60 

.27 

345 

.60 

.42 

816 

.60 

.62 

838 

.70 

.35 

335 

.70 

.51 

805 

.70 

.71 

828 

.80 

.44 

324 

.80 

.59 

794 

.80 

.79 

818 

.90 

.52 

314 

.90 

.68 

783 

.90 

.88 

.97808 

18.00 

14.60 

.97304 

23.00 

18.76 

.96772 

28.00 

22.96 

798 

.10 

.68 

294 

.10 

.84 

761 

.10 

23.04 

788 

.20 

.77 

283 

.20 

.92 

749 

.20 

.13 

778 

.30 

.85 

273 

.30 

19.01 

738 

.30 

.21 

768 

.40 

.94 

263 

.40 

.09 

726 

.40 

.30 

.97758 

18.50 

15.02 

.97253 

23.50 

19.17 

.96715 

28.50 

23.38 

748 

.60 

.10 

242 

.60 

.25 

704 

.60 

.47 

738 

.70 

.18 

232 

.70 

.34 

692 

.70 

.55 

728 

.80 

.27 

222 

.80 

.42 

681 

.80 

.64 

718 

.90 

.38 

211 

.90 

.51 

669 

.90 

.72 

.97708 

19.00 

15.43 

.97201 

24.00 

19.59 

.96658 

29.00 

23.81 

698 

.10 

.51 

191 

.10 

.67 

646 

.10 

.89 

688 

.20 

.59 

180 

.20 

.76 

635 

.20 

.98 

678 

.30 

.68 

170 

.30 

.84 

623 

.30 

24.06 

668 

.40 

.76 

159 

.40 

.93 

611 

.40 

.15 

.97658 

19.50 

15.84 

.97149 

24.50 

20.01 

.96600 

29.50 

24.23 

648 

.60 

.93 

139 

.60 

.09 

587 

.60 

.32 

638 

.70 

16.01 

128 

.70 

.18 

576 

.70 

.40 

628 

.80 

.09 

118 

.80 

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564 

.80 

.49 

618 

.90 

.18 

107 

.90 

.35 

553 

.90 

.57 

ALCOHOLS 


17 


In  the  writer's  laboratory  this  formula  has  given  fairly  satis- 
factory results  when  applied  to  temperatures  differing  but  little 
from  the  standard.  The  actual  weighing  of  the  pyknometer 
must  always  be  made  at  a  temperature  above  the  "  dew  point," 
but  a  pyknometer  which  provides  for  expansion  of  its  contents 
without  loss  may  be  filled  and  adjusted  at  the  standard  tem- 
perature and  then  allowed  to  stand  until  it  reaches  room 
temperature  before  weighing. 

TABLES.  —  DENSITY  OF  PURE  WATER  FREE  FROM  AIR  l 


Temperature 

Density 

Temperature 

Density 

4°C. 

1.0000000 

21°  C. 

0.9980233 

15 

0.9991266 

22 

0.9978019 

15.56 

0.9990415 

23 

0.9975702 

16 

0.9989705 

24 

0.9973286 

17 

0.9988029 

25 

0.9970770 

18 

0.9986244 

26 

0.9968158 

19 

0.9984347 

27 

0.9965451 

20 

0.9982343 

28 

0.9962652 

Refractometer  Method 

In  solutions  containing  only  alcohol  and  water,  such  as  the 
distillates  obtained  in  the  specific  gravity  method  above  de- 
scribed, the  percentage  of  alcohol  can  be  found  from  the  index 
of  refraction  as  well  as  from  the  specific  gravity.  The  recently 
introduced  "  immersion  refractometer  "  is  the  most  convenient 
form  of  apparatus  for  this  purpose.  Figure  4  shows  the  appara- 
tus complete  in  position  for  an  observation,  while  Fig.  5  shows 
the  positions  of  the  principal  parts. 

The  liquid  to  be  examined  is  placed  in  a  small  beaker  sur- 
rounded by  water  of  the  required  temperature,  usually  17.5° 
or  20°,  and  the  refractometer  is  suspended  from  the  wire  frame 

1  According  to  Chappius  (Bureau  international  des  Poids  et  Mesures,  Travaux 
et  Me"moires  XIII,  1907).     The  data  given  above  are  taken  from  the  table  pub- 
lished by  the  Bureau  of  Standards,  which  shows  density  for  each  tenth  of  a 
degree  from  0  to  41°  C.,  referred  to  water  at  4°  C.  as  unity, 
c 


18 


METHODS  OF  ORGANIC  ANALYSIS 


in  such  a  position  that  the  prism  is  immersed  in  the  liquid  to 
be  observed.  By  means  of  a  mirror  the  light  from  a  Avindow 
is  reflected  through  the  glass  bottom  of  the  water  bath  and 
upward  through  the  refractometer.  On  looking  downward 
through  the  ocular  Oc  and  the  telescope  of  the  refractometer 


FIG.  4.  —  General  view  of  immersion  refractometer. 
(Courtesy  of  Eimer  and  Amend.) 

one  observes  the  border  line  of  total  reflection,  the  upper  part 
of  the  field  of  vision  being  light  and  the  lower  part  shaded. 
A  scale,  marked  in  degrees  of  arbitrary  but  known  and  con- 
stant value,  extends  from  top  to  bottom  of  the  field  of  vision, 
and  the  position  of  the  border  of  the  shadow  upon  this  scale 
indicates  the  index  of  refraction.  The  index  of  refraction  cor- 


ALCOHOLS 


19 


responding  to  each  degree  of  the  immersion  refractometer  scale 
is  shown  in  Table  4. 

In  using  this  refractometer,  after  everything  is  in  position 
it  should  be  allowed  to  stand  ten  minutes  before  taking  obser- 
vations in  order  to  insure  uniformity  of  temperature.  The 


FIG.  5.  —  Sectional  view  of  immersion  refractometer. 
(Courtesy  of  Eimer  and  Amend.) 

adjustment  of  the  instrument  should  first  be  tested  by  taking 
reading  on  distilled  water.  The  index  at  R  should  stand  at 
5  and  the  ocular  Oo  should  be  focused  so  that  the  edge  of  the 
shadow  is  clearly  marked.  If  when  the  micrometer  screw  Z 
stands  at  zero  the  line  lies  between  two  of  the  scale  degrees,  its 


20 


METHODS   OF   ORGANIC   ANALYSIS 


TABLE  4. —  INDEX  OF  REFRACTION  FOR  EACH  DEGREE  ON  SCALE  OF 
THE  IMMERSION  REFRACTOMETER 


Scale  reading 

Index  of  refraction 

Scale  reading 

Index  of  refraction 

Scale  reading 

Index  of  refraction 

-5 

1.32539 

32 

1.33972 

69 

1.35352 

-4 

1.32578 

33 

1.34010 

70 

1.35388 

-3 

1.32618 

34 

1.34048 

71 

1.35425 

-2 

1.32657 

35 

1.34086 

72 

1.35461 

-1 

1.32696 

36 

1.34124 

73 

1.35497 

0 

1.32736 

37 

1.34162 

74 

1.35533 

1 

1.32775 

38 

1.34199 

75 

1.35569 

2 

1.32814 

39 

1.34237 

76 

1.35606 

3 

1.32854 

40 

1.34275 

77 

1.35642 

4 

1.32893 

41 

1.34313 

78 

1.35678 

5 

1.32932 

42 

1.34350 

79 

1.35714 

6 

1.32971 

43 

1.34388 

80 

1.35750 

7 

1.33010 

44 

1.34426 

81 

1.35786 

8 

1.33049 

45 

1.34463 

82 

1.35822 

9 

1.33087 

46 

1.34500 

83 

1.35858 

10 

1.33126 

47 

1.34537 

84 

1.35894 

11 

1.33165 

48 

1.34575 

85 

1.35930 

12 

1.33204 

49 

1.34612 

86 

1.35966 

13 

1.33242 

50 

1.34650 

87 

1.36002 

14 

1.33281 

51 

1.34687 

88 

1.36038 

15 

1.33320 

52 

1.34724 

89 

1.36074 

16 

1.33358 

53 

1.34761 

90 

1.36109 

17 

1.33397 

54 

1.34798 

91 

1.36145 

18 

1.33435 

55 

1.34836 

92 

1.36181 

19 

1.33474 

56 

1.34873 

93 

1.36217 

20 

1.33513 

57 

1.34910 

94 

1.36252 

21 

1.33551 

58 

1.34947 

95 

1.36287 

22 

1.33590 

59 

1.34984 

96 

1.36323 

23 

1.33628 

60 

1.35021 

97 

1.36359 

24 

1.33667 

61 

1.35058 

98 

1.36:394 

25 

1.33705 

62 

1.35095 

99 

1.36429 

26 

1.33743 

63 

1.35132 

100 

1.36464 

27 

1.33781 

64 

1.35169 

101 

1.36500 

28 

1.33820 

65 

1.35205 

102 

1.36535 

29 

1.33858 

66 

1.35242 

103 

1.36570 

30 

1.33896 

67 

1.35279 

104 

1.36605 

31 

1.33934 

68 

1.35316 

105 

1.36640 

ALCOHOLS 


21 


position  may  be  estimated  in  tenths  of  a  degree  by  the  eye  or 
the  micrometer  screw  Z  may  be  turned  until  the  line  of  the 
shadow  comes  exactly  to  a  scale  division.  .  The  latter  then  in- 
dicates the  whole  degrees  and  the  tenths  are  read  from  the 
scale  on  the  micrometer  screw.  This  method  is  usually  more 
accurate  than  estimating  the  tenths  by  the  eye  alone,  and  has 
the  advantage  that  one  may  quickly  turn  the  micrometer  screw 
back  to  zero  and  repeat  the  operation  as  often  as  desired,  finally 
averaging  the  readings.  With  water  the  reading,  depending 
upon  the  temperature,  should  be  as  follows : 


Temperature  C. 
Scale  reading 

17.5° 
15.0 

20° 
14.5 

21° 
14.25 

22° 
14.0 

23° 
13.75 

24° 
13.5 

25° 
13.25 

26° 
13.0 

27° 
12.7 

28° 
12.4 

29° 
12.1 

30° 
11.8 

Having  thus  tested  the  adjustment  of  the  instrument  by 
readings  in  water,  the  readings  in  the  alcoholic  solution  are 
taken  in  the  same  way.  The  percentages  by  weight  of  alcohol 
corresponding  to  the  scale  readings  at  17.5°  as  found  by  Acker- 
niann  and  Steinmann l  are  as  follows : 

TABLE  5.  —  PERCENTAGES   BY  WEIGHT  OF  ALCOHOL  CORRESPONDING  TO 
SCALE  READINGS  OF  ZEISS  IMMERSION  REFRACTOMETER  AT  17.5°  C. 

(ACKERMANN    AND    STEINMANN) 


Scale  reading 

Alcohol  per  cent 

Scale  reading 

Alcohol  per  cent 

15.0 

0.00 

19.5 

2.80 

15.5 

0.32 

20.0 

3.10 

16.0 

0.64 

20.5 

3.38 

16.5 

0.95 

21.0 

3.67 

17.0 

1.25 

21.5 

3.96 

17.5 

1.57 

22.0 

4.22 

18.0 

1.87 

22.5 

4.49 

18.5 

2.19 

23.0 

4.76 

19.0 

2.49 

23.5 

5.02 

For  tables  extending  to  higher  percentages  of  alcohol  (some- 
times expressed  as  percentage  by  volume  or  grams  per  100  cc.), 
1  Ztschr.  f.  der  gesamte  Brauwesen,  28  (1905). 


22  METHODS   OF   ORGANIC   ANALYSIS 

see  papers  by  Wagner,  Wagner  and  Schultze,  arid  Doroshevski 
and  Dvorzhanchik  among  references  given  at  the  end  of  this 
chapter.  The  table  given  by  the  last-named  authors  is  particu- 
larly noteworthy,  since  it  gives  the  actual  index  of  refraction 
for  alternate  percentages  of  alcohol  from  0  to  100  and  for  the 
temperatures  17.5°,  20°,  22°,  24°  C. 

For  a  fuller  description  of  the  immersion  refractometer  see 
Leach's  Food  Inspection  and  Analysis  (latest  edition)  or  the 
circulars  furnished  by  the  manufacturer  of  the  instrument. 

Boiling  Point  Method 

In  mixtures  of  alcohol  and  water  containing  no  appreciable 
amount  of  other  volatile  substances  and  only  small  quantities 
of  dissolved  solids,  the  difference  between  the  boiling  point  of 
the  mixture  and  that  of  pure  water  under  the  same  conditions 
gives  a  measure  of  the  percentage  of  alcohol  present.  For  the 
rapid  determination  of  alcohol  on  this  principle,  several  forms 
of  ebullioscope  have  been  devised.  The  liquid  to  be  tested  is 
boiled  under  a  reflux  condenser  while  a  thermometer  bulb  is 
fixed  just  above  the  surface  of  the  liquid  so  as  to  be  entirely 
surrounded  by  the  vapor.  The  more  common  technical  forms, 
such  as  those  of  Pohl  and  Kappeller,  have  scales  reading  per- 
centage of  alcohol  instead  of  thermometer  scales.  Water  is 
first  boiled  in  the  apparatus  and  the  scale  adjusted  so  that  the 
mercury  stands  at  zero.  If  then  the  water  be  removed  and  the 
sample  introduced  and  brought  to  boiling  under  the  same  baro- 
metric conditions,  the  point  reached  by  the  mercury  column 
shows  the  amount  of  alcohol  present. 

Wiley1  uses  a  delicate  differential  thermometer  with  an 
apparatus  similar  to  that  employed  for  the  determination  of 
molecular  weights  by  the  boiling  point  method.  Up  to  five 
per  cent  of  alcohol,  the  depression  of  the  boiling  point  is  said 
to  be  so  regular  that  the  results  are  entirely  satisfactory  for 
practical  work.  In  the  ebullioscopes  bearing  scales  graduated 
in  terms  of  alcohol,  the  variations  in  the  boiling  point  curve  at 

1  J.  Am.  Chem.  Soc.,  1896,  18,  1063. 


ALCOHOLS  23 

the  higher  percentages  are,  of  course,  allowed  for.  The  boiling 
point  method  is  very  rapid  and  gives  results  sufficiently  accu- 
rate for  many  purposes.  Reference  may  be  made  to  Vaubel  x 
for  a  general  discussion  of  methods  based  on  the  determination 
of  the  boiling  point  and  to  Freyer2  for  experimental  results  on 
the  influence  of  dissolved  solids  in  the  ebullioscopic  determina- 
tion of  alcohol. 

Oxidation  Method 

Under  suitable  conditions  ethyl  alcohol  can  be  quantitatively 
oxidized  to  acetic  acid  by  means  'of  potassium  dichromate  in 
the  presence  of  sulphuric  acid.  The  amount  of  alcohol  can 
then  be  ascertained  either  by  determining  the  amount  of  dichro- 
mate reduced3  or  by  distilling  and  titrating  the  acetic  acid 
formed.4  The  conditions  of  oxidation  must  be  carefully  regu- 
lated, and  as  a  rule  the  method  is  used  only  for  the  determina- 
tion of  very  small  amounts  of  alcohol,  the  specific  gravity 
method  being  preferable  for  the  examination  of  any  but  very 
dilute  solutions.  The  alcohol  must  of  course  be  separated  by 
distillation  from  any  other  oxidizable  matter  before  the  oxida- 
tion method  can  be  applied.  A  comparison  of  the  results  ob- 
tained by  oxidation  with  those  shown  by  the  specific  gravity 
method  may  be  useful  in  demonstrating  the  presence  of  homol- 
ogous alcohols. 

DETERMINATION  AND  IDENTIFICATION  OF  SMALL  AMOUNTS 

OF  ALCOHOL 

This  subject  has  been  studied  by  Bacon 5  with  special  refer- 
ence to  its  application  in  demonstrating  alcoholic  fermentation 
in  food  products.  „  Bacon  recommends  that  the  alcohol  be  con- 
centrated by  distillation  after  addition  of  salt  (which  as  noted 
above  results  in  the  alcohol  being  removed  with  a  smaller 

1  Quantitative  Bestimmung  organischer  Verbindungen.     Berlin,  1902. 

2  Z.  angew.  Chem.,  1896,  654. 

3  Hehner:  Analyst,  1887,  12,  25.     Benedict  and  Norris  :  J.  Am.  Chem.  Soc., 
1898,  20,  293.  *  r>upre :  J.  Chem.  Soc.,  1867,  20,  496. 

5  U.  S.  Dept.  Agriculture,  Bureau  of  Chemistry,  Circular  No.  74. 


24  METHODS   OF   ORGANIC   ANALYSIS 

amount  of  water)  after  which  the  distillate  is  examined  as  to 
density  and  index  of  refraction  as  well  as  by  chemical  tests  to 
'demonstrate  the  presence  of  alcohol.  In  a  typical  experiment 
given  by  Bacon,  1000  cc.  of  a  0.1  per  cent  solution  of  alcohol 
were  three  fourths  saturated  with  salt,  150  cc.  distilled  off, 
this  again  three  fourths  saturated  with  salt  and  25  cc.  distilled 
off.  This  distillate  showed  at  17.5°  a  refractometer  reading 
of  21.3  corresponding  to  3.84  per  cent  of  alcohol  in  the  dis- 
tillate or  0.096  per  cent  in  the  original  sample,  a  recovery  of 
96  per  cent  of  the  amount  present. 

Bacon  considers  that  if  the  refractometer  reading  and  the 
specific  gravity  indicate  the  same  percentage  of  alcohol  it  is 
almost  a  certainty  that  it  is  ethyl  alcohol  which  is  present,  and 
that  the  substance  under  examination  contains  the  percentage 
of  eth}d  alcohol  equivalent  to  these  constants.  As  a  further 
demonstration  Bacon  recommends  that  the  solution  containing 
the  alcohol  be  treated  with  a  slight  excess  of  paranitrobenzoyl 
chloride  and  an  equivalent  amount  of  sodium  hydroxide  and 
the  mixture  shaken  until  the  odor  of  the  acid  chloride  disap- 
pears, when  the  crystalline  ester  (readily  identified  by  its  melt- 
ing point  of  57°  C.1)  may  be  collected  and  weighed.  The 
yield  is  stated  to  be  70  to  90  per  cent  when  working  with 
small  quantities  of  alcohol.  If  the  paranitrobenzoyl  chloride 
is  not  available,  Bacon  suggests  benzoyl  chloride,  which  when 
used  in  the  same  way  yields  the  benzoic  acid  ethyl  ester  (ethyl 
benzoate),  which  may  be  weighed  (yield  said  to  be  nearly 
quantitative)  and  identified  by  its  odor  and  its  boiling  point, 
212°  C.,  using  for  the  determination  of  the  latter  the  method 
of  Smith  and  Menzies2;  and  that  in  addition  the  iodoform 
reaction  be  applied  since  the  only  benzoic  ester  having  an  odor 
similar  to  ethyl  benzoate  is  the  methyl  ester,  and  methyl  alcohol 
does  not  give  the  iodoform  reaction. 

DETECTION  AND  DETERMINATION  OF  METHYL  ALCOHOL 

The  great  difference  in  price  between  denatured  alcohol  and 
alcohol  which  has  been  subject  to  internal  revenue  tax  some- 

1  Ber.,  1905,  38,  620.  2  J.  Am.  Chem.  Soc.,  1910,  32,  897. 


ALCOHOLS  25 

times  results  in  the  substitution  of  denatured  alcohol  in  cases 
where  only  ethyl  alcohol  should  be  used.  This  is  detected  by 
demonstrating  the  presence  of  methyl  alcohol,  but  since  dena- 
tured alcohol  contains  only  one  part  of  methyl  to  ten  parts  of 
ethyl  alcohol,  it  is  evident  that  methods  to  be  useful  for  this 
purpose  must  be  applicable  to  the  detection  of  relatively  small 
amounts  of  methyl  alcohol  in  the  presence  of  relatively  large 
amounts  of  ethyl  alcohol. 

Many  methods  have  been  proposed  for  this  purpose.  Those 
which  probably  have  been  most  generally  used  are  based  upon 
the  oxidation  of  methyl  alcohol  to  formaldehyde  and  the  detec- 
tion of  the  latter  by  one  of  the  methods  described  in  the  next 
chapter.  In  the  methods  of  Mulliken  and  Scudder 1  and  of  the 
U.  S.  Pharmacopoeia  (Edition  of  1905,  p.  34)  the  oxidation  of 
methyl  alcohol  to  formaldehyde  is  accomplished  by  means  of 
a  copper  spiral  which  is  heated  until  covered  with  oxide  and 
then  plunged  into  the  liquid,  the  copper  being  reduced  and  the 
alcohol  partially  oxidized.  Yorisek2  prefers  to  oxidize  by 
means  of  chromic  acid;  and  Bacon  (loc.  cit.)  recommends  that 
5  to  8  grams  of  chromic  acid  be  added  to  100  cc.  of  the  aqueous 
methyl  alcohol  in  a  200  cc.  distilling  flask  and  the  first  10  cc. 
of  the  distillate  be  tested  for  formaldehyde.  The  results  of 
such  oxidation  methods  must  be  interpreted  with  caution,  since 
several  observers  have  reported  formaldehyde  among  the  prod- 
ucts of  oxidation  of  ethyl  alcohol. 

The  Association  of  Official  Agricultural  Chemists  have  pro- 
visionally adopted  the  methods  of  Trillat  and  of  Riche  and 
Bardy.  Trillat's  method3  is  based  upon  the  observation  that 
the  products  of  oxidation  of  ethyl  and  methyl  alcohol  combine 
with  dimethyl  aniline  to  form  bases  which  differ  in  their  color 
reactions.  The  method  of  Riche  and  Bardy4  depends  upon 
the  formation  of  methyl  aniline  violet. 

1  Am.  Chem.  J.,  1900,  24,  444.  2  J.  Soc.  Chem.  Ind.,  1909,  28,  823. 

3  Compt.    rend.,    1898,    127,   232.     Ann.   chim.    anal,    1899,    4,    42.      J. 
pharm.  chim.,  1899,  9,  372.    Analyst,  24,  13,  211,  212.     Leach's  Food  Inspec- 
tion and  Analysis,  2d  Ed.,  p.  750.    U.  S.  Dept.  Agr.,  Bur.  Chem.,  Bui.  107,  p.  99. 

4  Compt.  rend.,  1875,  80,  1076.     Allen's  Commercial  Organic  Analysis,  4th 
Ed.,  Vol.  I,  p.  98.     Leach's  Eood  Inspection  and  Analysis,  2d  Ed.,  p.  751. 
U.  S.  Dept.  Agr.,  Bur.  Chem.,  Bui.  107,  p.  99. 


26  METHODS   OF   ORGANIC   ANALYSIS 

The  Method  of  Leach  and  Lythgoe^-  depends  upon  the  fact 
that  although  ethyl  and  methyl  alcohol  in  solutions  have  very 
similar  densities,  they  differ  considerably  in  their  indices  of 
refraction.  This  difference  in  properties  is  utilized  both  in 
detecting  the  presence  of  methyl  alcohol  and  in  estimating  its 
amount  as  follows  : 

Submit  the  alcoholic  distillate  obtained  in  the  determination 
of  alcohol  to  refraction  with  the  immersion  refractometer  at 
exactly  20°  C.  and  note  the  reading.  If  on  reference  to  the 
table  the  refraction  shows  the  percentage  of  alcohol  agreeing 
with  that  obtained  from  the  specific  gravity  in  the  regular 
manner,  it  may  safely  be  assumed  that  no  methyl  alcohol  is 
present.  If,  however,  there  is  an  appreciable  amount  of 
methyl  alcohol,  the  low  refractometer  reading  will  at  once  in- 
dicate the  fact. 

Addition  of  methyl  to  ethyl  alcohol  decreases  the  refraction 
in  direct  proportion  to  the  amount  present ;  hence  the  quan- 
titative calculation  is  readily  made  by  interpolation  in  the 
table,  using  the  figures  for  pure  ethyl  and  methyl  alcohol  of 
the  same  alcoholic  strength  as  the  sample. 

Example :  Suppose  the  distillate  from  a  vanilla  extract  made 
up  to  the  original  volume  of  the  measured  portion  taken  for 
the  alcohol  determination  has  a  specific  gravity  of  0.97350, 
corresponding  to  18.38  per  cent  alcohol  by  weight,  and  has  a 
refraction  of  35.8  on  the  immersion  refractometer  at  20°.  By 
interpolation  in  the  refractometer  table  the  readings  of  ethyl 
and  methyl  alcohol  corresponding  to  18.38  per  cent  alcohol  are 
47.2  and  25.4,  respectively,  the  difference  being  21.8  ;  47.2- 
35.8  =  11.4  ;  (11. 4 -21.8)100  =  52.3,  showing  that  52.3  per  cent 
of  the  alcohol  present  is  methyl. 

The  Method  of  Thorpe  and  Holmes  2  is  probably  the  best  for 
the  quantitative  analysis  of  mixtures  of  ethyl  and  methyl 
alcohols  in  cases  in  which  the  immersion  refractometer  is  not 
available.  It  depends  upon  the  oxidation  of  the  mixture  of 

1  J.  Am.  Chem.  Soc.,  1905,  27,  964.    U.  S.  Dept.  Agr.,  Bur.  Chem.,  Cir.  29 
and  Bui.  107. 

2  J.  Chem.  Soc.,  1904,  85,  1. 


ALCOHOLS 


27 


TABLE  6.  —  SCALE  READINGS  ON  ZEISS  IMMERSION  REFRACTOMETER  AT 
20°  C.,  CORRESPONDING  TO  EACH  PER  CENT  BY  WEIGHT  OF  ETHYL 
AND  METHYL  ALCOHOLS  (LEACH  AND  LYTHGOE) 


4J          f 

<=-  ** 

III 

PHa,0 

Scale  readings 

Per  cent 
alcohol 
by  weight 

Scale  readings 

lit 

,?.£(>> 

••H   C3  ,O 

Scale  readings 

Per  cent 
alcohol 
bv  weight 

Scale  readings 

Methyl 
alcohol 

Ethyl 
alcohol 

Methyl 
alcohol 

Ethyl 
alcohol 

Methyl 
alcohol 

Ethyl 
alcohol 

Methyl 
alcohol 

Ethyl 
alcohol 

0 

14.5 

14.5 

26 

30.3 

61.9 

51 

39.7 

91.1 

76 

29.0 

101.0 

1 

14.8 

16.0 

27 

30.9 

63.7 

52 

39.6 

91.8 

77 

28.3 

100.9 

2 

15.4 

17.6 

28 

31.6 

65.5 

53 

39.6 

92.4 

78 

27.6 

100.9 

3 

16.0 

19.1 

29 

32.2 

67.2 

54 

39.5 

93.0 

79 

26.8 

100.8 

4: 

16.6 

20.7 

30 

32.8 

69.0 

55 

39.4 

93.6 

80 

26.0 

100.7 

5 

17.2 

22.3 

31 

33.5 

70.4 

56 

39.2 

94.1 

81 

25.1 

100.6 

6 

17.8 

24.1 

32 

34.1 

71.7 

57 

39.0 

94.7 

82 

24.3 

100.5 

7 

18.4 

25.9 

33 

34.7 

73.1 

58 

38.6 

95.2 

83 

23.6 

100.4 

8 

19.0 

27.8 

34 

35.2 

74.4 

59 

38.3 

95.7 

84 

22.8 

100.3 

9 

19.6 

29.6 

35 

35.8 

75.8 

60 

37.9 

96.2 

85 

21.8 

100.1 

10 

20.2 

31.4 

36 

36.3 

76.9 

61 

37.5 

96.7 

86 

20.8 

99.8 

11 

20.8 

33.2 

37 

36.8 

78.0 

62 

37.0 

97.1 

87 

19.7 

99.5 

12 

21.4 

35.0 

38 

37.3 

79.1 

63 

36.5 

97.5 

88 

18.6 

99.2 

13 

22.0 

36.9 

39 

37.7 

80.2 

64 

36.0 

98.0 

89 

17.3 

98.9 

14 

22.6 

38.7 

40 

38.1 

81.3 

65 

35.5 

98.3 

90 

16.1 

98.6 

15 

23.2 

40.5 

41 

38.4 

82.3 

66 

35.0 

98.7 

91 

14.9 

98.3 

16 

23.9 

42.5 

42 

38.8 

83.3 

67 

34.5 

99.1 

92 

13.7 

97.8 

17 

24.5 

44.5 

43 

39.2 

84.2 

68 

34.0 

99.4 

93 

12.4 

97.2 

18 

25.2 

46.5 

44 

39.3 

85.2 

69 

33.5 

99.7 

94 

11.0 

96.4 

19 

25.8 

48.5 

45 

39.4 

86.2 

70 

33.0 

100.0 

95 

9.6 

95.7 

20 

26.5 

50.5 

46 

39.5 

87.0 

71 

32.3 

100.2 

96 

8.2 

94.9 

21 

27.1 

52.4 

47 

39.6 

87.8 

72 

31.7 

100.4 

97 

6.7 

94.0 

22 

27.8 

54.3 

48 

39.7 

88.7 

73 

31.1 

100.6 

98 

5.1 

93.0 

23 

28.4 

56.3 

49 

39.8 

89.5 

74 

30.4 

100.8 

99 

3.5 

92.0 

24 

29.1 

58.2 

50 

39.8 

90.3 

75 

29.7 

101.0 

100 

2.0 

91.0 

25 

29-.7 

60.1 

ethyl  and  methyl  alcohols  under  such  conditions  that  the 
former  is  converted  into  acetic  acid  while  the  latter  is  com- 
pletely oxidized  to  carbon  dioxide  and  water.  The  total 
amount  of  alcohols  (estimated  as  ethyl  alcohol)  having  been 


28  METHODS  OF  ORGANIC  ANALYSIS 

determined  by  distillation  and  specific  gravity,  a  part  of  the 
distillate  is  mixed  with  water  in  such  proportions  that  50  cc. 
of  the  mixture  shall  contain  not  more  than  1  gram  of  methyl 
alcohol  nor  more  than  4  grams  of  ethyl  and  methyl  alcohols 
together.  Fifty  cubic  centimeters  of  this  mixture  are  intro- 
duced into  a  300-cc.  flask  having  a  tight  stopper  and  fitted 
with  a  funnel  and  side  tube,  20  grams  of  potassium  dichromate 
and  80  cc.  of  dilute  sulphuric  acid  (1 :  4)  added,  and  the  mix- 
ture allowed  to  remain  for  18  hours.  A  further  quantity  of  10 
grams  of  potassium  dichromate  and  50  cc.  of  sulphuric  acid 
mixed  with  an  equal  volume  of  water  are  now  added,  and  the 
Contents  of  the  flask  heated  to  the  boiling  point  for  about  10 
minutes,  the  evolved  carbon  dioxide  being  swept  out  of  the 
apparatus  by  a  current  of  air  and  collected  in  weighed  soda- 
lime  tubes.  Under  these  conditions  each  gram  of  ethyl  alco- 
hol was  found  to  yield  about  0.01  gram  of  carbon  dioxide. 
The  remaining  carbon  dioxide  found  is  calculated  as  being 
derived  from  the  complete  oxidation  of  methyl  aclohol. 

DETERMINATION  OF  AMYL  ALCOHOLS  OR  FUSEL  OIL 

The  amyl  alcohols  are  the  principal  constituents  of  fusel  oil, 
and  most  of  the  methods  proposed  for  the  determination  of 
fusel  oil  in  distilled  liquors  are  essentially  attempts  to  estimate 
the  amyl  alcohols.  In  order  to  separate  the  amyl  alcohols 
from  the  relatively  large  amounts  of  ethyl  alcohol  ordinarily 
present,  advantage  is  taken  of  the  fact  that  the  former  are 
much  more  soluble  in  chloroform  or  carbon  tetrachloride  than 
is  the  latter,  so  that  on  shaking  a  small  amount  of  chloroform 
or  carbon  tetrachloride  with  a  distillate  containing  ethyl  and 
amyl  alcohols  practically  all  of  the  amyl  alcohols  and  only 
a  little  of  the  ethyl  alcohol  is  extracted  by  the  chloroform  or 
the  carbon  tetrachloride. 

The  amount  of  amyl  alcohols  or  of  fusel  oil  is  usually  esti- 
mated either: 

1.  By  extracting  under  fixed  conditions  with  an  accurately 
known  volume  of  chloroform  and  estimating  the  fusel  oil  from 


ALCOHOLS  29 

the  increase  in  volume  of  the  chloroform  layer  (Roese's 
method). 

2.  By  extracting  with  carbon  tetrachloride,  oxidizing  the 
alcohols  of  the  extracted  fusel  oil  to  the  corresponding  acids 
by  means  of  potassium  dichromate,  and  distilling  and  titrating 
the  acids  thus  formed  (Allen-Marquardt  method). 

Both  of  these  methods  have  been  adopted  provisionally  by 
the  Association  of  Official  Agricultural  Chemists  and  are  given 
in  full  on  pp.  97-98,  Bui.  107  (Revised),  Bureau  of  Chemistry, 
U.  S.  Dept.  Agriculture. 

OFFICIAL  REQUIREMENTS  AS  TO  PURITY 
Ethyl  Alcohol 

The  U.  S.  Pharmacopoeia  in  addition  to  the  requirements 
above  given  and  a  test  which  is  supposed  to  show  methyl 
alcohol  if  more  than  two  per  cent  is  present,  prescribes  the  fol- 
lowing tests  of  purity  for  alcohol : 

It  should  not  affect  the  color  of  blue  or  red  litmus  paper  previously 
moistened  with  water. 

If  50  cc.  of  alcohol  be  evaporated  in  a  clean  vessel,  no  color  or  weighable 
residue  should  remain. 

If  10  cc.  of  alcohol  be  mixed  with  5  cc.  of  water  and  1  cc.  of  glycerin, 
and  the  mixture  allowed  to  evaporate  spontaneously  from  a  piece  of  clean, 
odorless  blotting  paper,  no  foreign  odor  should  become  perceptible  when  the 
last  traces  of  the  alcohol  leave  the  paper  (absence  of  fusel  oil  constitu- 
ents) . 

If  25  cc.  be  allowed  to  evaporate  spontaneously  in  a  porcelain  evaporat- 
ing dish,  carefully  protected  from  dust,  until  the  surface  of  the  dish  is 
barely  moist,  no  red  or  brown  color  should  be  produced  upon  the  addition 
of  a  few  drops  of  colorless,  concentrated  sulphuric  acid  (absence  of  amyl 
alcohol,  or  non-volatile  carbonizable,  organic  impurities,  etc.). 

If  10  cc.  of  alcohol  be  mixed  in  a  test  tube  with  5  cc.  of  potassium 
hydroxide  test  solution,  the  liquid  should  not  at  once  assume  a  yellow 
color  (absence  of  aldehyde  or  oak  tannin). 

If  20  cc.  of  alcohol  be  shaken  in  a  clean  glass-stoppered  vial  with  1  cc.  of 
silver  nitrate  test  solution,  the  mixture  should  not  become  more  than  faintly 
opalescent,  nor  acquire  more  than  a  faint  brownish  tint  when  exposed  for 
six  hours  to  diffused  daylight  (limit  of  organic  impurities,  amyl  alcohol, 
aldehyde,  etc.). 


30  METHODS  OF  ORGANIC  ANALYSIS 

Methyl  Alcohol 

The  following  requirements  have  been  established  by  the 
Bureau  of  Internal  Revenue  for  methyl  alcohol  to  be  used  in 
denaturing  grain  alcohol : 

The  methyl  alcohol  submitted  must  be  partially  purified  wood  alcohol 
obtained  by  the  destructive  distillation  of  wood.  It  must  conform  to  the 
following  analytical  requirements : 

1.  Color.  —  This  shall  not  be  darker  than  that  produced  by  a  freshly  pre- 
pared solution  of  2  cc.  of  tenth-normal  iodine  diluted  to  1000  cc.  with 
distilled  water. 

2.  Specific  Gravity.  —  It  must  have  a  specific  gravity  of  not  more  than 
0.830  at  60°  F.  (15.56°  C.),  corresponding  to  91°  of  Tralles'  scale. 

3.  Boiling  Point.  —  One  hundred   cubic   centimeters   slowly  heated  in  a 
flask  under  conditions  as  described  below  must  give  a  distillate  of  not  less 
than  90  cc.  at  a  temperature  not  exceeding  75°  C.  at  the  normal  pressure  of 
the  barometer  (760  mm.) .  One  hundred  cc.  of  wood  spirit  are  run  into  a  short- 
necked  copper  flask  of  about  180-200  cc.  capacity  and  the  flask  placed  on  an 
asbestos  plate  having  a  circular  opening  of  30  mm.  diameter.     In  the  neck  of 
this  flask  is  fitted  a  fractionating  tube  12  mm.  wide  and  170  mm.  long,  with 
a  bulb  just  1  centimeter  below  the  side  tube  which  is  connected  with  a  Lie- 
big's  condenser  having  a  water  jacket  not  less  than  400  mm.  long.     In  the 
upper  opening  of  the  fractionating  tube  is  placed  a  standardized  thermome- 
ter, so  adjusted  that  its  mercury  bulb  comes  in  the  center  of  the  bulb.     The 
distillation  is  conducted  in  such  a  manner  that  5  cc.  pass  over  in  one  minute. 
The  distillate  is  run  into  a  graduated  cylinder,  and  when  the  temperature 
of  75°  C.  has  been  reached  at  the  normal  barometric  pressure  of  760  mm.  at 
least  90  cc.  shall  have  been  collected. 

Should  the  barometer  vary  from  760  mm.  during  the  distillation,  1°  C. 
shall  be  allowed  for  every  variation  of  30  mm.  For  example,  at  770  mm. 
90  cc.  should  have  distilled  at  75.3°  C.,  and  at  750  mm.  90  cc.  should  have 
distilled  at  74.7°  C. 

4.  Miscibility  with  Water.  —  It  must  give  a  clear  or  only  slightly  opales- 
cent solution  when  mixed  with  twice  its  volume  of  water. 

5.  Acetone  Content.  —  It  must  contain  not  more  than  25  nor  less  than  15 
grams  per  100  cc.  of  acetone  and  other  substances  estimated  as  acetone  when 
tested  by  the  following  method  (Messinger). 

One  cubic  centimeter  of  a  mixture  of  10  cc.  wood  alcohol  with  90  cc.  of 
water  is  treated  with  10  cc.  of  double  normal  soda  solution.  Then  50  cc.  of 
tenth-normal  iodine  solution  are  added  while  shaking,  and  the  mixture  made 
acid  with  dilute  sulphuric  acid  three  minutes  after  the  addition  of  the 
iodine.  The  excess  of  iodine  is  titrated  back  with  tenth-normal  sodium 
thiosulphate  solution,  using  a  few  drops  of  starch  solution  for  an  indicator. 


ALCOHOLS  31 

From  15.5  to  25.8  cc.  of  tenth-normal  iodine  solution  should  be  used  by 
the  spirit.  The  solution  should  be  kept  at  a  temperature  between  15°  and 
20°  C. 

Calculation  : 

x  =  grams  of  acetone  in  100  cc.  of  spirit. 

y  =  number  of  cubic  centimeters  of  tenth-normal  iodine  solution  required. 
JV  =  volume  of  spirit  taken  for  titration. 


N 

6.  Esters.  —  It  should  contain  not  more  than  5  grams  of  esters  per  100  cc. 
of  spirit,  calculated  as  methyl  acetate  and  determined  as  follows  : 

Five  cubic  centimeters  of  wood  spirit  are  run  into  a  flask  and  10  cc.  normal 
sodium  hydroxide  free  from  carbonates  are  added,  and  the  flask  connected 
with  a  return  condenser  and  boiled  for  two  hours.  Instead  of  digesting  at 
boiling  temperature  the  flask  may  be  allowed  to  stand  overnight  at  room 
temperature  and  then  heated  on  a  steam  bath  for  thirty  minutes  with  an 
ordinary  tube  condenser.  The  liquid  after  digestion  is  cooled  and  titrated 
with  normal  sulphuric  acid,  using  phenolphthalein  as  an  indicator. 

Methyl  acetate,  in  grams  1  _  .074  x  cc.  of  normal  soda  required  x  100 
per  100  cc.  of  spirit       j  cc.  of  spirit  taken 

7.  Bromine  Absorption.  —  It  must  contain  a  sufficient  quantity  of  impurities 
derived  from  the  wood  so  that  not  more  than  25  cc.  nor  less  than  15  cc.  shall 
be  required  to  decolorize  a  standard  solution  containing  0.5  gram  of  bromine, 
as  follows  : 

The  standard  bromine  solution  is  made  by  dissolving  12.406  grams  of 
potassium  bromide  and  3.481  grams  of  potassium  bromate  (which  is  of 
tested  purity  and  has  been  dried  for  two  hours  at  100°  C.)  in  a  liter  of  water. 
Fifty  cubic  centimeters  of  the  standard  solution  containing  0.5  gram  of 
bromine  are  placed  in  a  glass-stoppered  flask  having  a  capacity  of  about  200 
cc.  This  is  acidified  by  the  addition  of  10  cc.  of  diluted  sulphuric  acid 
(1:4),  and  the  whole  shaken  and  allowed  to  stand  a  few  minutes.  The 
wood  alcohol  is  then  allowed  to  flow  slowly  into  the  mixture,  drop  by  drop, 
from  a  burette  until  the  color  is  entirely  discharged.  The  temperature  of 
the  mixture  should  be  20°  C. 

In  addition  to  the  above  requirements  the  methyl  alcohol  must  be  of  such 
a  character  as  to  render  the  ethyl  alcohol  with  which  it  is  mixed  unfit  for 
use  as  a  beverage. 

REFERENCES 


ALLEN  :    Commercial  Organic  Analysis,  Vol.  1. 

LEACH  :    Food  Inspection  and  Analysis. 

LUNGE  :    Chemisch-technische  Untersuchungsmethoden,  Bd.  III. 


32  METHODS  OF  ORGANIC  ANALYSIS 

MEYER  :   Analyse  und  Konstitutionsermittehmg  organischer  Verbindungen. 
MEYER-TINGLE  :  Determination  of  Radicles  in  Carbon  Compounds. 
MULLIKEN  :   Identification  of  Pure  Organic  Compounds,  Vol.  I. 
SCHMIDT  :   Ausfiihrliches  Lehrbuch  der  pharmaceutischen  Chemie,  Bd.  II, 

Abth.  I. 
U.  S.  Dept.  Agriculture,  Bureau  of  Chemistry,  Bui.  107  (Revised).     Official 

and  Provisional  Methods  of  Analysis. 
U.  S.  Dept.  Commerce  and  Labor,  Bureau  of  Standards,  Circular  No.  19, 

Standard  Density  and  Volumetric  Tables. 
U.  S.  Pharmacopoeia. 
VAUBEL  :   Die  physikalischen  und  chemischen  Methoden  der  quantitativen 

Bestimmung  organischer  Verbindungen. 
YOUNG  :   Fractional  Distillation. 


II 

1905.  LEACH  and  LYTHGOE  :    The  Detection  and  Determination  of  Ethyl 

and  Methyl  Alcohols  in  Mixtures  by  the  Immersion  Ref  ractom- 

eter.    /.  Am.  Chem.  Soc.,  27,  964. 
SCHIDROWITZ  and  KAYE  :   The  Determination  of  Higher  Alcohols  in 

Spirits.     Analyst,  30,  190. 
WINKLER:    (Preparation  of  Ethyl  Alcohol) .     Ber.,  38,  3612 ;  Analyst, 

31,  76. 

1906.  SCUDDER  and  RIGGS  :   The  Detection  of  Methyl  Alcohol.     J.  Am. 

Chem.  Soc.,  28,  1202. 

TOLMAN  and  TRESCOTT  :  Methods  for  the  Determination  of  Esters, 
Aldehydes,  and  Furfural  in  Alcoholic  Liquors.  J.  Am.  Chem. 
Soc.,  28,  1619. 

1907.  FLEISCHER  and  FRANK  :    (Rapid  Estimation  of  Alcohol  and  Ether  in 

their  Mixtures).     Chem.  Ztg.,  31,  665. 
SCHIDROWITZ  :   The  Estimation  of  Higher  Alcohols  ("  Fusel  Oil ") 

in  Distilled  Liquors.     J.  Am.  Chem.  Soc.,  29,  561. 
U.S.    Internal  Revenue  Regulations,  No.  30  —  Revised.     Concerning 

Denatured   Alcohol,    Central  Denaturing    Bonded  Warehouses, 

and  Industrial  Distilleries. 
WAGNER  and  SCHULTZE  :    (Estimation   of  Ethyl  Alcohol  with  the 

Zeiss  Immersion  Ref rac tome ter).     Z.  anal.  Chem.,  46,  508. 

1908.  ANDREWS:   The    Refractive    Indices    of    Alcohol-Water    Mixtures. 

/.  Am.  Chem.  Soc.,  30,  353. 

CRAMPTON  and  TOLMAN:  A  Study  of  the  Changes  taking  Place  in 
Whiskey  stored  in  Wood.  J.  Am.  Chem.  Soc.,  30,  98. 

DOROSHEVSKII  and  DVORZHANCHIK  :  Index  of  Refraction  of  Mix- 
tures of  Alcohol  and  Water.  J.  Russ.  Phys.-Chem.  Soc.,  40, 101 ; 
Chem.  Abs.,  2,  2181. 


ALCOHOLS  33 

DUDLEY:  Notes  on  the  Roese  Method  for  the  Determination  of 
Fusel  Oil,  and  a  Comparison  of  the  Results  by  the  Allen-Mar  - 
quardt  Method.  J.  Am.  Chem.  Soc.,  30,  1271. 

HINKEL  :  The  Detection  of  Small  Quantities  of  Methyl  Alcohol  in 
the  Presence  of  Ethyl  Alcohol.  Analyst,  33,  417. 

1909.  PLUCKER  :   Preparation  of  Pure  Ethyl  Alcohol.     Z.  Nahr.  Genussm., 

17,  454. 
TOLMAN  and  HILLYER  :   Methods  of   Analysis  of  Distilled  Spirits. 

U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  122,  p.  206. 
VORICEK  :   Detection  of  Methyl  Alcohol  in  Ethyl  Alcohol.     /.  Soc. 

Chem.  Ind.,  28,  823. 

1910.  WILEY  :   Manufacture   of  Denatured  Alcohol.     U.  S.  Dept.  Agricul- 

ture, Bur.  Chem.,  Bui.  130. 

1911.  BACON  :   Detection  and  Determination  of  Small  Quantities  of  Ethyl 

and  Methyl  Alcohol  and  of   Formic  Acid.     U.  S.   Dept.   Agri- 
culture, Bur.  Chem.,  Cir.  No.  74. 

GORE  :  An  Electrically  Controlled  Constant  Temperature  Water 
Bath  for  the  Immersion  Refractometer.  J.  Ind.  Eng.  Chem.,  3, 
506. 


CHAPTER  II 

Aldehydes 

THE  most  important  methods  for  the  detection  and  deter- 
mination of  aldehydes  are  based  upon  reactions  of  oxidation,  of 
condensation,  and  of  direct  addition.  In  this  chapter  the 
analytical  application  of  these  reactions  will  be  illustrated  by 
methods  for  formaldehyde,  benzaldehyde,  and  vanillin. 

The  readiness  with  which  aldehydes  undergo  oxidation  gives 
them  the  property  of  reducing  ammoniacal  silver  solution,  which 
is  the  basis  of  one  of  the  most  delicate  qualitative  tests  for  this 
group  of  compounds.  The  test  maybe  carried  out  as  follows:1 

Mix,  in  a  test  tube  previously  cleaned  with  hot  sodium  hy- 
droxide solution,  1  cc.  of  ammoniacal  silver  nitrate  solution 
(containing  one  part  of  silver  nitrate  in  ten  parts  of  ammonium 
hydroxide  of  0.923  sp.  gr.)  and  1  cc.  of  ten  per  cent  sodium 
hydroxide  solution.  Shake  the  mixture  in  the  tube  and  then 
allow  two  or  three  drops  of  the  solution  to  be  tested  to  flow 
slowly  down  the  moistened  glass  surface  into  the  reagent. 
Shake  and  allow  to  stand  cold  for  five  minutes.  Aldehydes 
(and  a  few  other  compounds  including  some  of  the  polyatomic 
alcohols)  cause  the  production  of  a  dark  brown  or  black  pre- 
cipitate or  mirror  of  metallic  silver.  This  reaction  is  given  by 
all  of  the  ordinary  aldehydes  of  the  fatty  series,  including  the 
aldose  carbohydrates,  but  not  by  all  aromatic  aldehydes. 

The  ammoniacal  silver  solution  and  the  sodium  hydroxide 
must  not  be  mixed  in  advance  and  must  always  be  kept  cool,  as 
a  dangerously  explosive  precipitate  is  apt  to  form  on  warming 

1  Noyes  and  Mulliken  :  Identification  and  Class  Reactions  of  Organic  Sub- 
stances. Mulliken  :  Identification  of  Pure  Organic  Compounds,  Vol.  I.,  p.  22. 

34 


ALDEHYDES  35 

or  on  long  standing.  The  use  of  a  mixture  of  sodium  hydroxide 
and  ammoniacal  silver  nitrate  (Tollens'  aldehyde  reagent)  makes 
the  test  more  delicate  than  when  the  ammoniacal  silver  solution 
is  used  alone. 

Alkaline  solutions  of  other  metals  are  reduced  by  many  alde- 
hydes, especially  on  boiling,  and  many  quantitative  methods  for 
individual  aldehydes  are  based  upon  the  determination  of  the 
amount  of  metal  reduced. 

Condensation  reactions,  especially  with  phenylhydrazine, 
hydroxylamine,  and  phenols,  are  often  used  for  the  detection  and 
sometimes  for  the  determination  of  aldehydes.  A  general  dis- 
cussion of  such  methods  will  be  found  in  the  works  of  Vaube 
and  of  Meyer.  Several  special  methods  will  be  described  in 
this  and  the  two  following  chapters. 

Of  the  addition  reactions  of  aldehydes,  that  with  bisulphite  is 
of  especially  wide  application.  On  shaking  a  liquid  aldehyde 
or  a  concentrated  solution  of  aldehyde  in  water  or  ether,  with 
an  equal  volume  of  strong  sodium  bisulphite  solution,  addition 
takes  place  with  the  formation  of  the  saturated  compound 
RCH(OH)SO3Na  which  usually  separates  as  a  white  crystalline 
precipitate.  Ketones  containing  the  CH3CO  group  also  give 
the  reaction.  A  negative  result  is  not  conclusive,  as  the  addi- 
tion product  may  be  too  soluble  to  appear  as  a  precipitate. 

According  to  Ripper,1  the  bisulphite  reaction  can  be  utilized 
for  the  determination  of  any  aldehyde  soluble  in  water  or  which 
can  be  brought  into  solution  by  a  small  amount  of  alcohol.  A 
one  half  per  cent  solution  of  the  aldehyde  is  mixed  with  twice 
its  volume  of  a  solution  of  potassium  bisulphite  of  known 
strength  (about  12  grams  per  liter),  and  after  15  minutes  the 
excess  of  bisulphite  is  determined  by  titration  with  iodine. 
Ripper  .applied  this  method  with  satisfactory  results  to  solu- 
tions of  formaldehyde,  acetaldehyde,  benzaldehyde,  and  vanillin. 

A  similar  addition  reaction  gives  rise  to  the  well-known  and 
delicate  "  fuchsin  test "  for  aldehydes.  This  test,  as  developed 
by  Mulliken,2  is  as  follows: 

1  Monatsh.  Chem.,  1900,  21,  1079. 

2  Identification  of  Pure  Organic  Compounds,  Vol.  I,  p.  15. 


36  METHODS  OF  ORGANIC  ANALYSIS 

To  prepare  the  fuchsin  aldehyde  reagent,  dissolve  0.2  gram 
of  rosanilin,  or,  if  the  free  base  cannot  be  obtained,  of  the  hydro- 
chloride  or  acetate,  in  10  cc.  of  a  freshly  prepared,  cold,  saturated 
aqueous  solution  of  sulphur  dioxide.  Allow  the  solution  to 
stand  until  all  signs  of  pink  disappear  and  it  becomes  colorless 
or  pale  yellow.  This  will  require  several  hours.  Then  dilute 
with  water  to  200  cc.  and  preserve  for  use  in  a  tightly  stoppered 
bottle. 

To  5  cc.  of  this  reagent  add  0.05  gram,  or  one  drop,  of  the 
substance  to  be  tested  (if  pure,  or  a  few  drops  if  in  solution). 
If  the  substance  is  a  liquid,  or  dissolves  in  the  reagent,  allow  to 
stand  two  minutes  and  observe  the  color.  If  the  substance  does 
not  dissolve,  shake  gently  for  two  minutes  and  then  observe  the 
color.  /  The  appearance  of  a  distinct  pink,  red,  purple,  or  blue 
coloration  indicates  the  presence  of  an  aldehyde/  The  test  to 
be  of  value  must  be  applied  under  carefully  regulated  conditions. 
The  reagent  is  reddened  by  alkalies  or  alkaline  salts  of  weak 
acids,  by  heating  or  by  long  exposure  to  air  at  ordinary  tem- 
perature. In  general,  the  test  as  here  described  distinguishes 
aldehydes  other  than  carbohydrates  from  the  latter  and  from  ke- 
tones.  A  few  acetals  show  the  reaction  through  being  partially 
hydrolyzed  to  aldehydes  under  the  conditions  of  the  test.  Ace- 
tone and  some  other  soluble  ketones  prepared  by  destructive 
distillation  gradually  redden  the  reagent  if  added  to  it  in  large 
quantity  or  if  allowed  to  remain  in  contact  for  a  number  of 
minutes ;  but  this  is  thought  to  be  due  chiefly,  if  not  wholly, 
to  the  presence  of  traces  of  aldehydes  or  acetals  (Mulliken). 

This  reaction  serves  for  the  detection  of  minute  quantities  of 
aldehydes  present  as  impurities  in  commercial  alcohol,  and  for 
the  colorimetric  estimation  of  aldehydes  in  distilled  liquors.1 

FORMALDEHYDE 

Formaldehyde  gas,  produced  by  the  partial  oxidation  of 
methyl  alcohol,  is  freely  soluble  in  water  and  is  most  com- 

1  Medicos :  Forschungsber.  uber  Lebensmittel,  1895,  1,  299.  Bui.  107,  Bur. 
Chem.,  U.  S.  Dept.  Agriculture.  Tolman  and  Trescott :  J.  Am.  Chem.  Soc., 
1906,  28,  1619. 


ALDEHYDES  37 

monly  handled  as  a  35  to  40  per  cent  aqueous  solution.  Such 
solutions  are  often  designated  formalin,  formol,  or  formal. 
More  dilute  solutions  are  sometimes  sold  as  food  preservatives 
under  fanciful  or  misleading  names. 

In  dilute  aqueous  solution,  formaldehyde  exists  in  the 
"  monomolecular "  state,  as  CH2O.  Such  solutions  do  not 
change  if  kept  at  ordinary  temperature  in  closed  vessels. 
When  an  aqueous  solution  is  concentrated  either  by  sponta- 
neous evaporation  or  by  heating,  a  white  flocculent  deposit 
appears.  If  the  solution  is  then  separated  from  the  deposit,  it 
is  found  to  contain  condensed  or  polymerized  formaldehyde.1 
The  material  which  deposits  from  a  concentrated  aqueous 
solution  of  formaldehyde  has,  after  drying,  the  composition 
(CH2O)6  •  H2O  to  (CH2O)8  -  H2O.2  It  is  amorphous,  soluble 
in  warm  water,  and  has  an  odor  resembling  that  of  formal- 
dehyde. The  paraformaldehyde  of  commerce  consists  essen- 
tially of  this  material. 

Metaformaldehyde  (oxymethylene,  "  trioxymethylene "), 
(CHgO),,.,  may  also  be  formed  by  evaporation  of  formaldehyde 
solutions.  By  prolonged  digestion  at  ordinary  temperature 
or  by  heating  for  a  short  time  at  130°-150°  with  a  large  excess 
of  water,  metaformaldehyde  passes  into  solution  and  into  the 
"  mono-molecular  "  form.  Polymeric  modifications  of  formal- 
dehyde in  aqueous  solution  resemble  closely  the  original  sub- 
stance in  its  behavior  toward  reagents,  so  that,  as  measured  by 
the  ordinary  methods,  a  solution  does  not  lose  strength  by  the 
partial  polymerization  of  the  formaldehyde  so  long  as  all  re- 
mains in  solution. 

Commercial  solutions  of  formaldehyde  commonly  contain 
methyl  alcohol  and  may  contain  small  amounts  of  any  of 
the  impurities  of  commercial  wood  spirit.  Solutions  of  the 
usual  strength,  from  35  to  40  per  cent.,  should  have  specific 
gravities  of  about  1.08  to  1.11  at  15°,  lower  figures  ordina- 

1  Tollens  and  Mayer  :  Ser.,  1888,  21,  1571,  3503.     Kraut,  Eschweiler,  and 
Grossmann:  Ann.  Chem.,  1890,  258,  103. 

2  Losekann  :    Chem.  Ztg.,  1890,  14,  1408.     Delephine:    Compt.  rend.,  1897, 
124,  1525.     Beilstein:  Organische  Chemie,  Erganzbd.,  I.,  467. 


38  METHODS  OF  ORGANIC  ANALYSIS 

rily  indicating  the  presence  of  excessive  amounts  of  methyl 
alcohol.1 

The  methods  given  in  this  chapter  for  the  detection  and  de- 
termination of  formaldehyde  refer  especially  to  the  examina- 
tion of  commercial  solutions  containing  only  such  impurities 
as  ordinarily  occur  in  crude  preparations  of  formaldehyde,  or 
substances  which  might  be  used  with  formaldehyde  in  preserva- 
tive mixtures.  The  examination  of  food  products  for  formalde- 
hyde will  be  discussed  in  connection  with  other  food  preserva- 
tives in  a  subsequent  chapter. 

If  a  solution  to  be  examined  contains  dissolved  solids  which 
interfere  with  the  direct  application  of  the  tests  as  described, 
it  can  be  acidified  with  a  small  excess  of  phosphoric  or  sulphuric 
acid,  distilled,  and  the  test  applied  to  the  distillate.  The 
latter,  however,  will  never  contain  all  of  the  formaldehyde, 
since  some  is  always  polymerized  and  left  as  paraformaldehyde 
in  the  distilling  flask. 

DETECTION  AND  IDENTIFICATION 

Resorcin  Test* 

Mix  one  drop  of  a  1  per  cent  aqueous  solution  of  resorcin 
with  1  cc.  of  a  dilute  aqueous  solution  (preferably  about  0.2  per 
cent)  of  the  aldehyde.  Allow  the  mixture  to  flow  gently  down 
the  side  of  an  inclined  test-tube  containing  3-5  cc.  of  pure  con- 
centrated sulphuric  acid  (or  incline  the  test  tube  containing  the 
mixture  and  pour  in  the  acid).  Impart  a  gentle  rotary  motion 
to  the  liquids  by  cautiously  swaying  the  lower  end  of  the  tube 
through  a  circle  about  a  decimeter  in  diameter,  in  such  a  man- 
ner as  not  to  cause  the  disappearance  of  the  two  layers.  If 
formaldehyde  is  present,  a  red  ring  slightly  tinged  with  violet 
will  soon  appear.  Above  this  ring  a  light  flocculent  precip- 

1  On  the  determination  of  methyl  alcohol  in  formaldehyde  solutions  see  — 
Duyk  :  Ann.  chim.  anal.,  1901,  6,  407  ;  J.  Chem.  Soc.,  1902,  82,  ii,  110.     Stri- 
trar  :    Z.  anal.  Chem.,  1904,  43,  401.     Gnehm  and  Kaufler:    Z.  angew.  Chem., 
1904,  17,  673;  1905,  18,  93.     Bamberger:  Ibid.,  1904,  17,  1246. 

2  Mulliken  and  Scuddrr :  Am.  Chem.  J.,  1900,  24,  451.     Mulliken  :  Identi- 
fication of  Pure  Organic  Compounds,  Vol.  I,  p.  24. 


ALDEHYDES  39 

itate,  at  first  nearly  white  on  its  upper  surface  and  red- violet 
beneath,  but  soon  changing  to  flocks  that  are  red  throughout, 
will  be  seen  suspended  in  the  aqueous  upper  layer. 

This  reaction  is  very  satisfactory  for  solutions  containing  one 
part  of  formaldehyde  in  100  to  5000  parts  of  solution  and  can 
be  detected  to  a  dilution  of  1  :  100,000.  A  similar  reaction  is 
obtained  if  phenol  is  used  instead  of  resorcin. 

Gallic  Acid  Test1 

Mix  0.2  cc.  of  a  saturated  solution  of  gallic  acid  in  pure 
ethyl  alcohol,  with  1  to  2  cc.  of  the  solution  to  be  tested,  and 
introduce  a  layer  of  concentrated  sulphuric  acid,  as  in  the 
resorcin  test.  In  the  presence  of  formaldehyde,  a  green  zone 
appears  at  the  line  of  contact  of  the  two  liquids.  This  gradu- 
ally changes  to  a  pure  blue  ring,  which,  in  the  case  of  pure 
aqueous  solutions  of  formaldehyde,  can  be  detected  without 
difficulty  at  a  dilution  of  1 :  500,000.  If  the  solution  tested 
contains  as  much  as  one  part  of  formaldehyde  in  20,000,  a  yel- 
lowish color  appears  immediately  at  the  line  of  contact  of  the 
two  liquids.  This  quickly  turns  green,  and  the  blue  color  de- 
velops both  above  and  below  the  green  zone.  If  other  sub- 
stances which  give  color  reactions  are  also  present,  the  upper 
layer  will  vary  in  color,  but  the  green  and  lower  blue  ring  will 
still  appear  beneath  (Mulliken  and  Scudder).  On  swaying 
the  tube,  or  allowing  it  to  stand  for  some  time,  the  blue  color 
spreads  throughout  the  zone  and  a  pure  blue  ring  is  usually 
obtained.  The  color  is  quite  permanent  and  apparently  quite 
characteristic,  no  other  substance  having  been  noted  as  giving 
the  blue  ring.  Acetaldehyde  tested  in  the  same  way  gives  a 
reddish  brown  coloration. 

Hydrochloric  Acid  and  Casein  Test* 

Mix  5  cc.  of  the  solution  to  be  tested  with  5  cc.  of  pure  milk 
in  a  porcelain  casserole,  add  10  cc.  of  concentrated  hydrochloric 

iBarbier  and  Jandrier :  Ann.  chim.  anal,  1,  325;  Abs.  Analyst,  1896, 
21,  295.  Mulliken  and  Scudder :  Am.  Chem.  J.,  1900,  24,  444. 

2  Leach  :  Ann.  Kept.  Mass.  State  Board  of  Health,  1897,  558 ;  1899,  699. 


40  METHODS   OF   ORGANIC   ANALYSIS 

acid  containing  0.002  gram  of  ferric  chloride,  and  heat  slowly 
over  a  free  flame  nearly  to  boiling,  meanwhile  giving  the  cas- 
serole a  rotary  motion  to  break  up  the  curd.  -A  violet  colora- 
tion indicates  formaldehyde.  According  to  Leach,  various 
aldehydes  give  color  reactions  under  this  treatment,  but  form- 
aldehyde alone  shows  the  unmistakable  violet  coloration.  This 
test  is  especially  useful  for  the  detection  of  formaldehyde  in 
milk,  and  will  be  more  fully  discussed  in  that  connection. 

Meihylene-di-ft-naphthol  Test 

Since  formaldehyde  is  frequently  sold  under  other  names,  its 
identification  by  some  method  independent  of  the  above  color 
reactions  may  be  a  matter  of  importance.  In  such  cases  the 
following  test  given  by  Mulliken l  will  be  useful. 

Place  in  a  test  tube  3  drops  of  a  30  to  40  per  cent,  or  10 
drops  of  a  10  per  cent,  solution  of  the  formaldehyde,  3  cc.  of 
dilute  alcohol  (1 :  2),  0.04  to  0.06  gram  /3-naphthol,  and  3  to  5 
drops  of  concentrated  hydrochloric  acid.  Boil  gently  until 
the  liquid  becomes  filled  with  an  abundant  precipitate  of  small 
white  needles.  Filter  while  hot.  Wash  with  1  cc.  of  dilute 
alcohol  (1:2).  Boil  the  precipitate  with  4  cc.  of  dilute  alcohol 
(1 :  1).  (It  is  not  necessary  that  all  should  dissolve.)  Cool 
and  filter  off  the  precipitate.  Wash  with  1  cc.  of  dilute  alcohol 
(1:1).  Dry  on  porous  tile  in  a  warm  place  and  determine 
the  melting  point. 

Methylene-di-/3-naphthol,  the  product,  forms  white  needles, 
which,  when  the  temperature  in  the  neighborhood  of  the  melt- 
ing point  is  raised  at  the  rate  of  1°  in  15  seconds,  begin  to  turn 
brown  at  180°.  It  melts  with  decomposition  to  a  red-brown 
liquid  at  189°-192°  (uncorr.). 

DETERMINATION  BY  OXIDATION 

lodimetric  Method2 

This  method  depends  upon  the  oxidation  of  formaldehyde  to 
formic  acid  by  means  of  iodine  in  alkaline  solution.  Two  atoms 

1  Identification  of  Pure  Organic  Compounds,  Vol.  I,  p.  24. 
2Romijn:  Z.  anal.  Chem.,  1897,  36,  18.     Williams:   J.  Am.   Chem.  Soc., 
1905,  27,  596. 


ALDEHYDES  41 

of  iodine  oxidize  one  molecule  of  formaldehyde,  and  the  excess  of 
iodine  is  liberated  by  acidulation  and  determined  by  titration 
with  sodium  thiosulphate. 

CH20  +  12  +  H20  =  CH202  +  2  HI. 

Reagents.  —  Standard  solutions  of  iodine  and  sodium  thiosul- 
phate, preferably  about  tenth-normal.  Approximately  normal 
solutions  of  sodium  hydroxide  and  hydrochloric  acid. 

Determination.  —  Dilute  a  weighed  portion  of  the  sample  with 
a  known  quantity  of  water  so  as  to  obtain  a  solution  containing 
0.5  to  1  per  cent  of  actual  formaldehyde.  Mix  10  cc.  of  this 
solution  with  25  cc.  normal  sodium  hydroxide  and  add  from  a 
burette  50  to  75  cc.  of  tenth-normal  iodine  solution  or  enough 
to  assure  an  excess  of  iodine  as  shown  by  the  permanent  yellow- 
color  of  the  solution.  Shake  or  stir  thoroughly  and  after  ten 
minutes  add  35  cc.  normal  hydrochloric  acid  and  'titrate  with 
sodium  thiosulphate  in  the  usual  way,  using  starch  solution  as 
indicator.  At  the  same  time  determine  the  strength  of  the 
iodine  in  terms  of  thiosulphate  solution  and  from  the  amount 
of  iodine  consumed  in  oxidizing  the  formaldehyde  calculate  the 
weight  of  the  latter  in  the  10  cc.  taken  for  the  determination. 

Notes.  —  Under  the  conditions  given  the  oxidation  of  form- 
aldehyde is  rapid  and  complete  but  the  method  is  applicable 
only  in  the  absence  of  all  other  substances  capable  of  consuming 
iodine  under  these  conditions.  Other  aldehydes,  acetone,  and 
alcohol  cause  high  results,  the  latter  probably  through  absorb- 
ing iodine  with  the  formation  of  iodoform. 

In  the  absence  of  interfering  compounds,  the  method  is  very 
satisfactory,  even  for  solutions  containing  only  0.1  per  cent 
of  formaldehyde.  Variations  in  the  excess  of  iodine  added 
have  no  appreciable  influence  upon  the  results. 

Hydrogen  Peroxide  Method1 

In  this  method,  formaldehyde  is  oxidized  to  formic  acid  by 
means  of  hydrogen  peroxide  in  the  presence  of  a  known  amount 
of  alkali. 


1  Blank  and  Finkenbeiner  :  J5er,  1898,  31,  2979.     Haywood  and  Smith:  J. 
Am.  Chem.  Soc.,  1905,  27,  1183.    Bui.  107,  Bar.  Chem.,  U.  S.  Dept.  Agriculture. 


42  METHODS  OF  ORGANIC  ANALYSIS 

The  excess  of  alkali,  over  that  required  to  combine  with  the 
formic  acid  produced,  is  determined  by  titration. 

The  method  is  here  described  as  used  by  the  Association  of 
Official  Agricultural  Chemists. 

Reagents.  —  Normal  solutions  of  sodium  hydroxide  and  sul- 
phuric acid.  Neutral 1  3  per  cent  solution  of  hydrogen  peroxide. 
Solution  of  purified  litmus  as  indicator. 

Determination.  —  Measure  50  cc.  of  normal  sodium  hydroxide 
into  a  500-cc.  Erlenmeyer  flask,  add  50  cc.  hydrogen  peroxide 
solution,  then  3  grams  of  the  formaldehyde  solution.  Place  a 
funnel  in  the  neck  of  the  flask  and  stand  it  on  a  steam  bath  for 
5  minutes,  shaking  occasionally  during  this  time.  Remove, 
wash  funnel  with  water,  cool  to  room  temperature,  and  titrate 
excess  of  alkali  with  normal  acid,  using  litmus  as  indicator. 
Each  molecule  of  sodium  hydroxide  which  has  been  consumed 
(deducting  the  amount  required  to  neutralize  any  free  acid 
which  the  peroxide  solution  or  the  original  solution  of  form- 
aldehyde may  have  contained)  represents  one  molecule  of 
formaldehyde  oxidized  to  formic  acid. 

Notes.  —  The  use  of  exactly  3  grams  of  formaldehyde  is  not 
essential,  but  the  exact  weight  must  of  course  be  known.  It  is 
convenient  to  use  a  weighing  bottle  containing  a  small  pipette, 
measure  out  about  3  cc.  and  obtain  the  weight  by  difference. 

Acetaldehyde  is  partially  oxidized  under  the  same  conditions. 
Its  presence,  therefore,  causes  high  results,  but  not  so  high  as 
by  the  iodimetric  method.  The  results  are  not  influenced  by 
the  presence  of  paraldehyde,  acetone,  or  ethyl  or  methyl  alcohol. 
Commercial  formalin  containing  only  traces  of  acetone  or 
acetaldehyde  should  show  the  same  percentage  of  formaldehyde 
by  the  peroxide  as  by  the  iodimetric  method. 

DETERMINATION  BY  CONDENSATION  REACTIONS 

Several  of  the  condensation  reactions  of  formaldehyde  have 
been  utilized  for  its  quantitative  determination.  One  of  the 

1  If  all  available  peroxide  is  acid,  the  acidity  must  be  determined  by  titration, 
using  litmus  as  indicator,  and  allowed  for  in  calculating  the  amount  of  alkali 
consumed  in  the  formaldehyde  determination. 


ALDEHYDES  43 

oldest  and  best-known  methods  is  based  upon  the  fact  that 
formaldehyde  and  ammonia  when  mixed  in  not  too  dilute 
solution  condense  to  form  hexamethylene  tetramine : 

6  CH2O  +  4  NH4OH  =  N4(CH2)6  +  10  H2O. 

If  a  known  amount  of  ammonia  is  used,  the  determination 
of  the  excess  shows  the  amount  of  formaldehyde  originally 
present. 

Legler's  Ammonia  Method**- 

Weigh  about  1.5  grams  of  the  solution  containing  30  to  40 
per  cent  formaldehyde,  or  an  equivalent  amount  of  a  more 
dilute  solution,  into  a  250-cc.  glass-stoppered  flask  or  bottle. 
Add  100  cc.  of  fifth-normal  ammonia  solution ;  stopper  tightly 
at  once ;  mix  and  allow  to  stand  overnight  at  room  tempera- 
ture. Standing  for  two  or  three  days  does  no  harm,  provided 
the  stopper  fits  so  tightly  as  to  prevent  any  loss  of  ammonia. 
Finally,  add  a  very  small  amount  of  rosolic  acid  as  indicator 
ahd  titrate  the  excess  of  ammonia  with  standard  sulphuric  acid. 
Calculate  the  quantity  of  formaldehyde  originally  present  from 
the  amount  of  ammonia  consumed  in  condensing  with  it  ac- 
cording to  the  equation  given  above. 

Notes  and  Precautions.  —  In  order  to  prevent  loss  of  ammonia 
during  the  determination,  the  flask  or  bottle  must  be  tightly 
closed,  the  stopper  being  coated  with  vaseline  if  necessary. 
For  the  same  reason  the  excess  of  ammonia  should  be  titrated 
quickly  after  opening  the  flask.  Normal  or  half-normal 
ammonia  is  commonly  recommended  for  this  method,  but  the 
fifth-normal  solution  is  less  likely  to  lose  strength  and  has  been 
found  by  Williams  to  give  as  complete  reactions  as  the  stronger 
solutions.  In  titrating  the  excess  of  ammonia  the  end  reaction 
is  usually  unsatisfactory,  especially  when  the  solution  is  highly 
colored  by  the  indicator.  Two  drops  of  a  freshly  prepared  0.1 
per  cent  solution  of  rosolic  acid  have  been  found  sufficient. 

The  results  are  not  affected   by   the   presence   of  acetone, 

iLegler:  Ber.,  1883,  16,  1333.  Smith  :  J.  Am.  Chem.  Soc.,  1903,  25,  1028. 
Williams :  loc.  cit. 


44  METHODS   OF   ORGANIC   ANALYSIS 

methyl  or  ethyl  alcohol,  paraldehyde,  or  benzaldehyde.  Acetal- 
dehyde  reacts  with  ammonia  and  thus  causes  high  results  if 
present  in  the  formaldehyde  solution. 

This  method  was  formerly  much  used  in  analysis  of  com- 
mercial formalin,  but  on  account  of  the  tendency  toward  low 
results  it  is  now  generally  displaced  by  the  hydrogen  peroxide 
method. 

DETERMINATION  BY  ADDITION  REACTIONS 

The  general  addition  reaction  of  aldehydes  with  bisulphites 
has  been  used  quantitatively  by  Ripper,  as  already  noted.  For 
the  determination  of  formaldehyde,  however,  the  reaction  with 
potassium  cyanide  has  been  found  especially  useful. 

Potassium   Cyanide  Method^- 

On  mixing  aqueous  solutions  of  formaldehyde  and  potassium 
cyanide  an  addition  product  is  formed,  which,  according  to 
Romijn,  is  probably  the  potassium  compound  of  oxyacetonitril  : 

CH2O  +  KCN  =  CH2OK .  CN. 

The  addition  product  reduces  silver  nitrate  in  alkaline  solu- 
tion, but  has  no  effect  in  the  presence  of  an  excess  of  nitric  acid. 
If,  therefore,  the  formaldehyde  to  be  tested  be  mixed  with  a 
known  solution  of  potassium  cyanide,  the  latter  being  in  excess, 
and  the  mixture  added  to  a  standard  solution  of  silver 
nitrate  acidulated  with  nitric  acid,  only  the  excess  of  potassium 
cyanide  reacts  with  the  silver  nitrate.  The  amount  of  formal- 
dehyde originally  present  is  shown  by  the  quantity  of  potassium 
cyanide  consumed  in  the  formation  of  the  addition  product. 
The  details  of  the  method  as  here  given  are  nearly  identical 
with  those  originally  recommended  by  Romijn. 

Reagents.  —  Tenth-normal  solutions  of  silver  nitrate  and 
ammonium  thiocyanate.  A  solution  of  potassium  cyanide  6.2 
grams  per  liter..  Saturated  solution  of  ferric  ammonium 
sulphate.  Nitric  acid  1.32  sp.  gr.  (50  per  cent). 

1  Romijn:  Z.  anal.  Chem.,  1897,  36,  18.  Smith:  loc.  cit.  Williams:  Joe. 
cit. 


ALDEHYDES  45 

Determination.  —  (1)  Measure  15  cc.  tenth-normal  silver 
nitrate  into  a  100-cc.  flask,  add  6  to  8  drops  of  the  nitric  acid 
and  10  cc.  of  the  cyanide  solution ;  shake,  dilute  to  the  mark, 
mix  thoroughly,  and  filter  through  a  dry  paper.  Titrate  50  cc. 
of  the  nitrate  with  tenth-normal  ammonium  thiocyanate,  using 
5  cc.  of  the  ferric  solution  as  indicator.  The  strength  of  the 
silver  and  of  the  thiocyanate  solutions  being  known,  this  titra- 
tion  shows  the  strength  of  the  cyanide. 

(2)  Dilute  the  sample  until  it  contains  about  1  per  cent  of 
formaldehyde,  mix  10  cc.  of  this  dilute  solution  with  35  cc.  of 
the  cyanide  solution,  and  rinse  the  mixture  into  another  portion 
of  15  cc.  tenth-normal  silver  nitrate,  acidulated  with  6  to  8 
drops  of  the  nitric  acid  and  contained  in  a  100-cc.  flask ;  shake 
and  determine  the  excess  of  silver  by  means  of  thiocyanate  in 
the  same  way  as  before.  Twice  the  difference  between  the  two 
titrations  (since  only  half  the  liquid  was  used  in  each  case) 
represents  the  amount  of  cyanide  consumed  by  the  formaldehyde. 
If  the  thiocyanate  solution  is  exactly  tenth-normal,  twice  the 
difference  (in  cubic  centimeters)  between  the  two  titrations, 
multiplied  by  0.0030Q2,  gives  the  weight  of  formaldehyde  (in 
grams)  in  the  portion  taken  for  the  determination. 

Notes.  — This  method  is  applicable  to  very  dilute  solutions  of 
formaldehyde,  larger  volumes  being  used  in  place  of  the  10  cc. 
called  for  in  the  above  directions.  Smith  obtained  accurate 
results  upon  a  solution  containing  0.01  per  cent. 

Ethyl  and  methyl  alcohols,  acetone,  benzaldehyde,  and  paral- 
dehyde  do  not  interfere.  Acetaldehyde  causes  high  results  if 
allowed  to  stand  for  some  time  in  contact  with  the  cyanide 
solution,  but  if  the  formaldehyde  solution  is  added  to  the 
cyanide  and,  after  mixing,  poured  at  once  into  the  silver  nitrate 
solution,  the  presence  of  acetaldehyde  does  not  influence  the 
results.  With  commercially  pure  solutions  of  formaldehyde  in 
water  Romijn  and  Smith  obtained  concordant  results  by  the 
iodimetric  and  cyanide  methods.  Williams  obtained  concord- 
ant results  by  the  ammonia  and  the  cyanide  methods,  which 
were  slightly  lower  than  those  obtained  by  the  oxidation 
methods. 


46  METHODS   OF   OKGANIC   ANALYSIS 

BENZALDEHYDE 

Many  methods  have  been  advanced  for  the  determination  of 
benzaldehyde.  Among  these  the  methods  based  on  reactions 
with  phenylhydrazine  and  its  derivatives,  and  with  neutral 
sulphite,  are  worthy  of  special  notice  in  that  they  are  fairly 
accurate  and  serve  to  illustrate  the  analytical  application  of 
fairly  general  aldehyde  reactions. 

PHENYLHYDRAZINE  METHOD 

By  simple  condensation  benzaldehyde  and  phenylhydrazine 
yield  an  insoluble  derivative  which  may  be  collected,  washed, 
dried,  and  weighed. 

C6H5CHO  +  C6H5NH.NH2=C6H5CH  :  N  .  NH  .  C6H5  +  H2O. 

This  method,  as  developed  by  Denis  and  Dunbar1  and  adopted 
by  the  Association  of  Official  Agricultural  Chemists,2  is  as 
follows  : 

Determination  of  Benzaldehyde  in  Almond  Extract  (Denis  and 

Dunbar) 

Reagent.  —  Add  1.5  cc.  of  glacial  acetic  acid  to  20  cc.  of 
water  and  mix  with  1  cc.  of  phenylhydrazine. 

Manipulation. — Measure  out  two  portions  of  10  cc.  each  of 
the  extract  in  300  cc.  Erlenmeyer  flasks  and  add  10  cc.  of  the 
reagent  to  one  flask  and  15  cc.  to  the  other.  Allow  to  stand 
in  a  dark  place  overnight,  add  200  cc.  of  water,  and  filter  on  a 
weighed  Gooch  crucible  having  a  thin  felt  of  asbestos.  Wash 
first  with  cold  water,  finally  with  10  cc.  of  10  per  cent  alcohol, 
and  dry  for  three  hours  in  a  vacuum  oven  at  70°  C.,  or  to  con- 
stant weight  over  sulphuric  acid.  If  the  duplicate  results  do 
not  agree,  repeat  the  determination,  using  a  larger  quantity  of 
the  reagent. 

NEUTRAL  SULPHITE  METHOD 

Benzaldehyde,  in  common  with  many  other  aldehydes,  reacts 
with  neutral  sodium  sulphite  in  such  a  way  that  there  results 

1  J.  Ind.  Eng.  Chem.,  1907,  1,  256. 

2  U.  S.  Dept.  Agr.,  Bur.  Chem.,  Bui.  137,  pp.  74,  121. 


ALDEHYDES  47 

the  aldehyde-bisulphite  addition  product  and  sodium  hydroxide, 
thus: 

C6H6CHO  +  Na2SO3  +  H2O  ==  C6H5CH(OH)SO3Na  +  NaOH. 

The  sodium  hydroxide  formed  is  titrated  and  furnishes  a 
measure  of  the  amount  of  benzaldehyde  which  was  present. 

The  details  of  this  method  as  adopted  in  the  U.S.  Pharma- 
copoeia are  as  follows : 

Assay  of  Benzaldehgde  (  U.  S.  Pharmacopoeia) 

Introduce  into  a  150-cc.  flask  10  cc.  purified  kerosene,  note 
the  exact  weight,  add  12  drops  of  benzaldehyde,  and  again  note 
the  weight;  add  20  cc.  of  water,  6  drops  of  phenolphthalein 
solution,  and  neutralize  exactly  by  the  addition  of  tenth-normal 
sodium  hydroxide,  shaking  thoroughly.  Then  add  from  a 
burette,  gradually,  a  solution  of  sodium  sulphite  (1  in  5), 
alternating  with  half-normal  hydrochloric  acid  from  a  second 
burette,  until  10  cc.  of  the  sodium  sulphite  solution  have  been 
added,  and  enough  half-normal  hydrochloric  acid  to  maintain 
the  neutrality  of  the  mixture ;  after  adding  a  few  drops  of 
phenolphthalein  solution  and  shaking  the  flask  frequently, 
allow  it  to  stand  two  hours  to  insure  a  permanent  condition  of 
neutrality,  and  then  note  the  volume  of  half -normal  acid  used. 
Carry  out  a  blank  test  identical  with  the  foregoing  except 
that  the  benzaldehyde  is  omitted  and  note  the  amount  of  acid 
consumed. 

From  the  difference  in  volume  of  the  half-normal  acid  used 
in  the  two  cases,  calculate  the  amount  of  benzaldehyde  which 
reacted  with  sulphite  according  to  the  equation  given  above. 

For  discussion  of  the  application  of  the  neutral  sulphite  method 
to  other  aldehydes  see  the  papers  of  Burgess1  and  Sadtler2. 

When  this  method  is  applied  to  aldehydes  which  have  an 
ethylene  linkage,  there  may  occur  a  further  reaction  with 
addition  of  bisulphite  at  this  point  as  well  as  at  the  carbonyl 
group  and  a  correspondingly  increased  liberation  of  sodium 
hydroxide. 

1  Analyst,  29,  78.  2  J.  Am.  Chem.  Soc.,  27,  1321. 


48  METHODS   OF   ORGANIC   ANALYSIS 

VANILLIN 

In  the  case  of  vanillin,  the  sulphite  method  appears  to  be 
inapplicable,  and  in  this  laboratory  the  bisulphite  method  has 
given  results  somewhat  too  low.1  Better  results  have  been 
obtained  by  the  alkalimetric  method  of  Welmans,2  and  by  the 
Hanus3  method  of  condensation  with  p.  bromphenylhydrazine : 

C6H3(OH)(OCH3)CHO  +  C6H4(Br)NH  •  NH2  = 
C6H3  (OH)(OCH3)  CH:  NH  .  NHC6  H4  Br  +  HaO. 

Twenty-five  cubic  centimeters  of  a  water  solution  containing 
0.5  to  1  per  cent  of  vanillin  are  treated  with  75  cc.  of  a  hot 
water  solution  containing  0.5  to  0.75  gram  of  p.  bromphenyl- 
hydrazine. At  the  conclusion  of  the  precipitation  the  mixed 
liquid  should  be  at  about  50°  ;  the  precipitate  is  allowed  to 
stand  5  hours,  filtered  on  a  Gooch  crucible,  washed  with  hot 
water  till  washings  show  no  precipitation  nor  distinct  color- 
ation with  silver  nitrate,  dried  at  95°-100°  to  constant  weight, 
and  weighed.  With  pure  vanillin  solutions  this  method  gave 
nearly  theoretical  results.1 

REFERENCES 


ALLEN  :  Commercial  Organic  Analysis. 

LUNGE  :  Chemisch-technische  Untersuchungsmethoden. 

MEYER  :  Analyse  und  Konstitutioiisermittelung  organise  her  Verbindungen. 

MULLIKEN  :  Identification  of  Pure  Organic  Compounds,  Vol.  I. 

VAUBEL  :  Quantitative  Bestimmung  organischer  Verbindungen. 

II 

1904.  BURGESS  :  Estimation  of  Aldehydes  and  Ketones  in  Essential  Oils. 

Analyst,  29,  78. 

1905.  HANUS  :  Ueber  eine   quantitative   Bestimmung  des  Vanillins.     Z. 

Nahr.  Genussm.,  10,  585. 

HAYWOOD  and  SMITH  :  A  Study  of  the  Hydrogen  Peroxide  Method 
of  Determining  Formaldehyde.     J.  Am.  Chem.  Soc.,  27,  1183. 

1  Determinations  by  B.  G.  Feinberg,  not  yet  published. 
*Pharm.  Ztg.,  1898,  34,  634  ;  Vaubel,  II,  88. 
8Z.  Nahr.  Genussm.,  3,  531  ;  10,  585. 


ALDEHYDES  49 

SADTLER  :  A  Fuller  Study  of  the  Neutral  Sulphite  Method  for  Deter- 
mining Some  Aldehydes  and  Ketones  in  Essential  Oils.  /. 
Am.  Chem.  Soc.,  27,  1321. 

WILLIAMS  :  A  Study  of  Methods  for  the  Determination  of  Formalde- 
hyde. J.  Am.  Chem.  Soc.,  27,  596. 

WINTON  and  BAILEY  :  The  Determination  of  Vanillin,  Coumarin, 
and  Acetanilid  in  Vanilla  Extract.  J.  Am.  Chem.  Soc.,  27,  719. 

1906.  CHASE  :  A  Method  for  the  Determination  of  Citral  in  Lemon  Oils  and 

Extracts.     J.  Am.  Chem.  Soc.,  28,  1472. 

1907.  DOBY:  (Comparison    of   Methods  for  Determination  of   Formalde- 

hyde).    Z.  angew.  Chem.,  20,  353. 

1908.  WOODMAN  and  LYFORD  :  The  Colorimetric  Estimation  of  Benzalde- 

hyde  in  Almond  Extracts.     J.  Am.  Chem.  Soc.,  30,  1607. 

1909.  DENIS   and   DUNBAR:  Determination   of   Benzaldehyde  in  Almond 

Flavoring  Extract.    J.  Ind.  Eng.  Chem.,  1,  256. 


CHAPTER   III 

Carbohydrates  —  General  Methods 

THE  carbohydrates  include  the  simple  sugars  (monosaccha- 
rides)  and  the  substances  which  can  be  converted  into  simple 
sugars  by  hydrolysis.  The  monosaccharides  are  aldehyde  alco- 
hols or  ketone  alcohols,  each  molecule  containing  a  carbonyl 
group,  either  as  such  or  in  tautomeric  form,  and  several  hydroxyl 
groups,  one  of  the  latter  being  adjacent  to  the  carbonyl  group. 

The  purpose  of  this  chapter  is  to  outline  the  more  important 
general  methods  and  analytical  properties  of  the  following  car- 
bohydrates: 

Monosaccharides:  Hexoses  —  Dextrose  (d.  glucose),  Levulose 
(d.  fructose),  Galactose,  Mannose;  Pentoses — Xylose,  Arabin- 
ose. 

Disaccharides :   Sucrose,  Lactose,  Maltose. 

Trisaccharide:  Raffinose. 

Polysaccharides :  Starch,  Dextrin,  Glycogen,  Galactan,  Cellu- 
lose, Pentosans. 

OCCURRENCE   AND  RELATIONS 

Monosaccharides  (glucoses,  glycoses,  monoses)  have  the  com- 
position (CHgO).,.1  and  are  called  tetroses,  pentoses,  hexoses, 
etc.,  according  to  the  number  of  carbon  atoms  in  the  molecule. 
Only  pentoses  and  hexoses  are  of  sufficient  practical  importance 
to  call  for  consideration  in  connection  with  ordinary  methods 
of  analysis.  The  pentoses  do  not  occur  free  in  nature  but 

1  This  statement  docs  not  apply  to  the  methyl  derivatives  now  frequently 
classified  as  monosaccharides. 

50 


CARBOHYDRATES  —  GENERAL  METHODS        51 

are  met  by  the  analyst  as  products  of  the  hydrolysis  of  the 
pentosans.  The  hexoses  include  all  of  the  monosaccharides 
of  present  commercial  importance  and  all  whose  biological 
relations  have  been  thoroughly  studied. 

Dextrose  (d.  glucose,  grape  sugar,  starch  sugar,  diabetic 
sugar,  ordinary  glucose)  is  widely  distributed  in  nature,  occur- 
ring especially  in  fruits  and  plant  juices,  often  mixed  with 
other  sugars.  It  is  a  normal  constituent  of  blood  and  is  the 
form  of  carbohydrate  ordinarily  found  in  the  urine  in  diabetes 
or  glycosuria.  With  the  exception  of  the  pentosans  and 
galactan,  all  of  the  di-,  tri-,  and  polysaccharides  mentioned 
above  yield  dextrose  on  hydrolysis. 

Levulose  (d.  fructose,  fruit  sugar)  occurs  with  dextrose  in 
plant  juices  and  especially  in  fruits  and  honey.  It  is  also  a 
product  of  the  hydrolysis  of  sucrose  and  of  raffinose. 

Galactose  does  not  occur  free ;  but  as  a  product  of  hydrolysis 
of  lactose,  raffinose,  and  the  galactans  it  is  of  considerable 
analytical  importance. 

Mannose  also  is  not  found  free,  but  has  been  detected  among 
the  products  of  hydrolysis  of  the  insoluble  carbohydrate  matter 
of  a  number  of  thick-walled  vegetables  tissues,  nut  shells,  etc., 
and  of  several  Japanese  vegetables. 

The  disaccharides  considered  here  are  all  hexo-bioses 
(C12H22On). 

Sucrose  (saccharose,  cane  sugar)  is  widely  distributed  in  the 
vegetable  kingdom,  being  found  in  considerable  quantity,  gen- 
erally mixed  with  dextrose  and  levulose,  in  the  fruits  and 
juices  of  many  plants.  The  most  important  sources  of  sucrose 
are  the  sugar  beet,  the  sugar  and  sorghum  canes,  and  the  sugar 
maple.  A  molecule  of  sucrose  yields  on  hydrolysis  one  mole- 
cule each  of  dextrose  and  levulose.  The  hydrolysis  of  sucrose 
is  often  called  "  inversion  "  and  the  resulting  mixture  of  equal 
parts  dextrose  and  levulose  is  known  as  "invert  sugar." 

Lactose  (lactobiose,  milk  sugar)  occurs  in  the  milk  of  most 
mammals,  constituting  usually  from  4  to  7  per  cent  of  the 
fresh  secretion.  Lactose  crystallizes  with  one  molecule  of 
water  which  it  retains  on  drying  at  room  temperature  over 


52  METHODS   OF   ORGANIC   ANALYSIS 

sulphuric  acid  or  on  heating  in  the  air  at  100°,  but  loses  at 
about  130°.  A  molecule  of  lactose  yields  on  hydrolysis  one 
molecule  each  of  dextrose  and  galactose. 

Maltose  (malt  sugar)  is  formed  from  starch  by  the  action  of 
diastatic  enzymes  and  is  therefore  an  important  constituent  of 
germinating  cereals,  malt,  malt  extract,  and  beer  wort.  It  is 
also  formed  as  an  intermediate  product  when  starch  is  hydro- 
lyzed  to  dextrose  by  boiling  with  dilute  mineral  acids,  as  in  the 
manufacture  of  commercial  glucose.  Maltose  crystallizes  with 
one  molecule  of  water,  which  it  loses  on  heating  in  the  air  at 
100°.  Each  molecule  of  maltose  yields  two  molecules  of  dex- 
trose on  hydrolysis. 

The  only  trisaccharide  of  practical  importance  is  raffinose 
(C18H32O16),  also  called  meletriose  and  formerly  melitose  or 
gossypose.  It  occurs  in  cotton  seed  and  in  small  quantity  in 
the  germs  of  various  other  seeds  including  wheat  and  barley. 
Sugar  beets,  especially  if  unhealthy  or  injured,  sometimes 
contain  raffinose  in  sufficient  quantity  to  affect  the  refining 
process.  Raffinose  crystallizes  with  five  molecules  of  water  in 
needles  or  slender  prisms  and  has  a  marked  influence  upon  the 
crystallization  of  the  cane  sugar  present.1  Raffinose  loses  its 
water  of  crystallization  at  100°.  On  hydrolysis  it  yields  one 
molecule  each  of  dextrose,  levulose,  and  galactose.  Partial 
hydrolysis  results  in  the  formation  of  levulose  and  the 
disaccharide,  melibiose. 

Starch  (C6H10O5)Z  is  the  most  important  of  the  polysac- 
charides,  being  the  principal  form  of  carbohydrate  in  grains 
and  most  other  edible  seeds,  as  well  as  in  potatoes  and  other 
tubers.  It  is  the  main  product  of  the  assimilation  process 
and  the  principal  reserve  carbohydrate  of  most  green  plants. 
Commercially  it  is  of  great  importance  as  a  constituent  of 
foods,  as  the  source  of  dextrin,  maltose,  and  commercial  glucose, 
and  as  the  principal  raw  material  of  many  of  the  fermentation 
industries.  Starch  constitutes  over  one  half  of  the  solid  matter 
of  all  ordinary  cereals  and  about  three  fourths  of  the  total  solids 
in  potatoes.  Starch  granules  of  different  plants  vary  in  size 

1  Stone  and  Baird:  J.  Am.  Chem.  Soc.,  1897,  19,  116. 


CARBOHYDRATES  —  GENERAL  METHODS         53 

and  structure  so  that  in  most  cases  the  source  of  a  starch  which 
has  not  been  altered  by  heat,  ferments,  or  chemical  reagents  can 
be  determined  by  microscopical  examination.  All  starches 
yield  dextrose  only,  as  the  final  product  of  complete  hydrolysis. 

Dextrins,  (C6H10O5)X  or  (C6H10O5)X  •  H2O,  are, formed  from 
starch  by  the  action  of  enzymes,  acids,  or  heat.  Small  amounts 
of  dextrin  are  found  in  normal,  and  larger  amounts  in  germi- 
nating, cereals.  Malt  diastase  acting  upon  starch  in  fairly 
concentrated  solution  yields  usually  about  one  part  of  dextrin 
to  four  of  maltose.  During  acid  hydrolysis,  dextrin  is  formed 
as  an  intermediate  product  between  soluble  starch  and  maltose. 
Commercial  dextrin,  the  principal  constituent  of  "  British 
gum,"  is  obtained  by  heating  starch,  either  alone  or  with  a 
small  amount  of  dilute  acid. 

Glycogen,  (C6H10O5)X  or  perhaps  (C6H10O5)X .  H2O,  is  the 
principal  carbohydrate  of  the  animal  organism,  being  found 
in  small  quantity  in  the  muscles  and  more  abundantly  in  the 
liver  of  all  well-nourished  animals.  It  is  a  white  amorphous 
powder  intermediate  in  properties  between  starch  and  dextrin 
and  is  sometimes  called  animal  starch.  The  determination  of 
glycogen  is  often  important  in  physiological  investigations  and 
is  sometimes  useful  in  distinguishing  horseflesh  from  beef,  the 
latter  containing  usually  less  than  0.7  per  cent  of  glycogen, 
the  former  often  two  or  three  times  this  amount.  On  com- 
plete hydrolysis  glycogen  yields  only  dextrose. 

Galactans,  amorphous  polysaccharides  yielding  galactose  on 
hydrolysis,  occur  in  small  quantity  in  many  plants  and  in 
relative  abundance  in  the  seeds  of  the  legumes  where  they 
largely  replace  starch  as  reserve  carbohydrate.  Since  the 
galactans  are  readily  hydrolyzed  by  hot  dilute  acids  and  are 
digested  by  some  of  the  diastatic  enzymes,  it  is  probable  that 
galactan  has  been  reported  as  starch  in  many  analyses. 

Cellulose  occurs  in  the  cell  walls  of  all  vegetable  tissues. 
The  term  is  sometimes  applied  to  the  whole  of  the  fiber  which 
is  unattacked  by  boiling  dilute  acids  and  alkalies,  but  should  be 
restricted  to  that  constituent  of  the  fiber  which  is  of  a  true 
carbohydrate  nature.  "  Normal "  cellulose,  such  as  is  derived 


54  METHODS   OF   ORGANIC   ANALYSIS 

from  cotton  and  flax  fibers,  yields  dextrose  on  hydrolysis. 
A  few  celluloses  have  been  found  to  yield  mannose  or  a 
pentose  (probably  xylose)  in  addition  to  dextrose  (Tollens). 

Pentosans,  anhydrides  of  arabinose  and  xylose,  are  the  prin- 
cipal constituents  of  the  vegetable  gums,  araban  occurring  espe- 
cially in  the  soluble  gums  such  as  cherry  gum  and  gum  arable, 
xylan  in  the  so-called  wood  gum  of  fibrous  tissues  such  as  wood, 
straw,  vegetables,  and  the  outer  portion  of  the  cereal  grains. 
The  wheat  grain,  for  example,  contains  3  to  5  per  cent  of 
pentosan,  which  in  the  milling  process  is  largely  left  in  the 
bran.  The  so-called  patent  flour  obtained  from  the  interior  of 
the  grain  contains  hardly  any  pentosan,  while  the  breakfast 
cereals  and  the  so-called  entire  wheat  and  graham  flours  have 
usually  about  as  much  as  the  original  grain. 

SOLUBILITIES 
IN  WATER 

Of  the  carbohydrates  mentioned  above,  all  except  the  poly- 
saccharides  are  crystallizable  compounds  dissolving  in  water 
to  form  clear  solutions.  Milk  sugar  dissolves  in  six  parts  of 
water  at  ordinary  temperature;  all  of  the  other  members  are 
more  freely  soluble.  Among  the  polysaccharides,  dextrin, 
glycogen,  and  some  of  the  galactans  and  pentosans  are  soluble ; 
starch,  cellulose,  some  of  the  galactans,  and  most  of  the  pento- 
sans of  ordinary  food  materials  are  insoluble  in  cold  water. 
Glycogen  gives  a  strongly  opalescent  solution,  which  is  not 
cleared  by  repeated  filtration  but  loses  its  opalescence  on  the 
addition  of  a  little  potassium  hydroxide  or  acetic  acid.  On 
heating  with  water,  starch  grains  swell  and  finally  gelatinize 
with  the  formation  of  "  starch  paste."  Different  starches  vary 
considerably  in  the  temperature  at  which  they  gelatinize  and 
in  the  physical  properties  of  the  paste  produced.  Thin  starch 
pastes  can  be  filtered  through  paper,  but  almost  always  leave 
some  gelatinous  residue  upon  the  filter.  Pastes  containing  only 
a  few  hundredths  of  one  per  cent  of  starch  become  clear  on 
boiling  and  can  be  filtered  without  loss.  Water-soluble  starch 


CARBOHYDRATES  —  GENERAL  METHODS         55 

can  be  prepared1  by  chemical  treatment  and  is  sometimes  found 
in  natural  products,  for  example  in  immature  grains. 

Cellulose  and  the  ordinary  pentosans  of  foods  and  fibers  are 
insoluble  in  water  and  not  gelatinized  by  boiling. 

IN  ALCOHOL  AND  ETHER 

Levulose  is  soluble  in  5  parts  of  cold  absolute  alcohol  and  is 
somewhat  soluble  in  mixtures  of  ether  and  strong  alcohol.  The 
other  monosaccharides  are  sparingly  soluble  in  cold  alcohol,  in- 
soluble in  ether,  and  practically  insoluble  in  the  alcohol-ether 
mixture.  Dextrose  is  much  more  readily  soluble  in  hot  than 
in  cold  alcohol ;  100  parts  of  90  per  cent  alcohol  dissolve  about 
2  parts  dextrose  at  18°,  about  22  parts  at  boiling  temperature. 

The  di-,  tri-,  and  polysaccharides  are  insoluble  in  ether.  Di- 
and  trisaccharides  are  less  soluble  in  alcohol  than  is  dextrose. 
Lactose  is  practically  insoluble  in  alcohol,  even  when  the  latter 
is  diluted  to  60  per  cent. 

Sucrose  is  much  more  readily  soluble  in  diluted  than  in  con- 
centrated alcohol.  According  to  Scheibler  : 

100  parts  90  per  cent  alcohol  dissolve    0.9  parts  at  14° ;  2.3  parts  at  40° 

100  parts  80  per  cent  alcohol  dissolve    6.6  parts  at  14° ;  13.3  parts  at  40° 

100  parts  70  per  cent  alcohol  dissolve  18.8  parts  at  14° ;  31.4  parts  at  40° 

100  parts  60  per  cent  alcohol  dissolve  33.9  parts  at  14° ;  49.9  parts  at  40° 

100  parts  50  per  cent  alcohol  dissolve  47.1  parts  at  14° ;  63.4  parts  at  40° 

Sucrose  dissolves  in  about  80  parts  of  boiling  absolute  alcohol. 

All  of  the  polysaccharides  are  insoluble  in  alcohol.  Those 
which  are  soluble  in  water  can  be  precipitated  from  their 
aqueous  solutions  by  the  addition  of  strong  alcohol. 

IN  ACIDS  AND  ALKALIES 

Among  the  carbohydrates  which  are  insoluble  in  water, 
separations  can  sometimes  be  made  by  the  use  of  acid  or  alka- 
line solutions. 

Cellulose  is  soluble  in  concentrated  sulphuric  acid,  but  in- 

1  Lintner  :  J.  prakt.  Chem.,  1886,  [2]  34,  381.  Wroblewski :  Z.  physiol. 
Chem.,  1898,  24,  173.  Vaubel :  II.,  500. 


56  METHODS  OF  ORGANIC  ANALYSIS 

soluble  in  any  ordinary  aqueous  solution  of  acid  or  alkali.  It 
dissolves  in  Schweitzer's  reagent  (aqueous  ammonia  saturated 
with  cupric  hydroxide)  to  a  viscous  solution,  from  which  it  is 
precipitated  by  neutralization  with  acid. 

Starch  is  insoluble  in  Schweitzer's  reagent  or  in  solutions  of 
ammonia.  Treated  with  dilute  aqueous  solutions  of  sodium  or 
potassium  hydroxide,  starch  swells,  gelatinizes,  and  becomes 
soluble.  It  can  be  completely  precipitated  from  such  a  solution 
by  neutralizing  with  acetic  acid  and  adding  strong  alcohol. 
Starch  is  not  affected  by  dilute  solutions  of  alkalies  in  strong 
alcohol.  By  diluted  solutions  of  strong  acids  starch  is  first 
dissolved,  then  hydrolyzed.  Some  weak  organic  acids  dissolve 
starch  with  little  if  any  hydrolysis.  Boiling  water  containing 
1  per  cent  of  salicylic  acid  dissolves  starch  to  an  opalescent  so- 
lution which  niters  much  more  readily  than  a  corresponding 
starch  paste  made  with  water  alone. 

The  pentosans  of  foods  and  fibers — so-called  wood  gums  con- 
sisting mainly  of  xylan  —  are  insoluble  in  dilute  ammonia  or  in 
Schweitzer's  reagent,  largely  soluble  in  dilute  aqueous  solutions 
of  sodium  or  potassium  hydroxide  and  in  cold  dilute  acids. 
From  such  solutions  the  pentosan  is  precipitated  by  alcohol. 
Boiling  dilute  mineral  acids  dissolve  and  hydrolyze  pentosans 
almost  as  readily  as  starch.  Hence  pentosans  have  frequently 
been  reported  as  starch  when  the  latter  has  been  estimated  by 
direct  hydrolysis  with  acid  and  determination  of  the  resulting 
glucose. 

REACTIONS  WITH  ACIDS 

All  carbohydrates  on  treatment  with  moderately  strong 
hydrochloric  or  sulphuric  acid  yield  furfural,  which  is  probably 
the  cause  of  the  color  reaction  with  a-naphthol  described  below. 
Hexacarbohydrates  form  only  small  amounts  of  furfural  and 
relatively  large  amounts  of  levulinic  acid.  Pentacarbohy- 
drates  yield  large  amounts  of  furfural  and  no  levulinic  acid. 
In  all  cases,  however,  there  is  more  or  less  formation  of 
"  humus  "  and  other  by-products. 

The  following  is  widely  used  as  a  general  qualitative  test  for 
carbohydrates. 


CARBOHYDRATES  —  GENERAL   METHODS  57 

MOLISCH'S  «-NAPHTHOL  REACTION  l 

Mix  2  to  3  cc.  of  the  very  dilute  solution  to  be  tested  with 
2  or  3  drops  of  a  15  per  cent  solution  of  a-naphthol  in  alcohol 
or  chloroform,  incline  the  tube,  and  pour  in  carefully  2  to  3  cc. 
of  pure  concentrated  sulphuric  acid.  In  the  presence  of  carbo- 
hydrate, a  violet  zone  appears  quickly  and  spreads  by  diffusion. 
If  the  solution  tested  contains  more  than  a  few  milligrams  of 
carbohydrate,  it  quickly  blackens  and  on  dilution  with  water 
gives  a  dull  violet  precipitate. 

A  similar  test  with  thymol  gives  a  crimson  or  carmine-red 
solution  which  soon  becomes  turbid. 

FURFURAL  TEST  FOR  PENTOSES  AND  PENTOSANS 

Place  the  substance  to  be  tested  in  an  Erlenmeyer  flask,  add 
hydrochloric  acid  (1.06  sp.  gr.),  and  boil.  Lay  over  the  mouth 
of  the  flask  a  small  filter  paper  moistened  with  a  solution  of 
anilin  acetate.2  If  the  vapor  escaping  from  the  flask  contains 
more  than  traces  of  furfural,  a  bright  red  coloration  appears. 
A  test  with  sucrose  or  pure  starch  will  show  the  amount  of 
color  to  be  expected  from  hexacarbohydrates. 

If  the  reaction  obtained  from  the  unknown  substance  is 
much  stronger  than  from  sucrose  or  starch,  the  presence  of 
pentoses  or  pentosans  is  indicated.  A  few  other  substances 
such  as  glycuronic  acid  and  oxycellulose  have  been  found  to 
yield  considerable  amounts  of  furfural,  but  these  rarely  occur 
in  sufficient  quantities  to  require  consideration. 

DETERMINATION  OF  PENTOSES  AND  PENTOSANS 

Under  carefully  regulated  conditions  the  yield  of  furfural 
from  either  xylose  or  arabinose,  or  from  the  corresponding 
anhydride,  is  nearly  constant.  If  the  furfural  is  distilled  and 
collected,  its  amount  can  be  estimated  by  adding  anilin  acetate 

1  Molisch :  Monatsh.  Chem.,  1886,  7,  198.     Udranszky :  Z.  physiol.  Chem., 
1888,  12,  355,  377.    Tollens  :  Handbuch  der  Kohlenhydrate,  II,  101.     Mulli- 
ken  :  Identification  of  Pure  Organic  Compounds,  I,  26. 

2  Prepared  by  mixing  equal  volumes  of  anilin  and  50  per  cent  acetic  acid. 


58  METHODS  OF  ORGANIC  ANALYSIS 

and  comparing  the  red  color  with  that  shown  by  furfural  solu- 
tions of  known  strength.1  This  method  is  delicate,  but  gives 
only  approximate  results.  Unless  the  quantity  is  very  small, 
it  should  be  determined  gravimetrically. 

Furfural  forms  sparingly  soluble  condensation  products  with 
a  number  of  bases  and  phenols.  Phenylhydrazine  and  phloro- 
glucin  have  been  principally  used  as  precipitants.2  The 
Association  of  Official  Agricultural  Chemists  has  adopted 
provisionally  the  phloroglucin  method.3 

LEVTJLINIC  ACID  REACTION  OF  HEXACARBOHYDRATES 

According  to  Wehmer  and  Tollens,4  all  hexacarbohydrates 
yield  levulinic  acid  in  sufficient  quantity  for  identification  when 
treated  as  follows  : 

Heat  5  to  20  grams  of  the  substance  with  100  cc.  of  hydro- 
chloric acid  (1.10  sp.  gr.)  for  18  hours  on  a  boiling  water  bath; 
filter  the  solution,  shake  with  ether  to  extract  the  levulinic  acid, 
and  convert  the  latter  into  the  zinc  or  silver  salt  for  identifica- 
tion. As  this  method  is  purely  qualitative  and  is  not  often 
used  in  ordinary  analytical  work,  reference  must  be  made  to 
the  original  paper  for  details  of  manipulation. 

OXIDATION  BY  NITRIC  ACID 

By  heating  with  moderately  strong  nitric  acid,  xylose  and 
arabinose  are  oxidized,  each  yielding  a  trioxyglutaric  acid, 
COOH(CHOH)3COOH,  while  aldoses  of  the  hexose  group 
yield  the  corresponding  acids,  COOH(CHOH)4COOH,  sac- 

1  This  colorimetric  method  is  also  used  for  the  estimation  of  furfural  in  dis- 
tilled liquors. 

2  Barbituric  acid  has  recently  been  recommended  as  preferable  to  phenyl- 
hydrazine  and  phloroglucin  as  a  precipitant  for  furfural,     linger  :  Dissertation, 
Munich,  1904.    Jager  and  Unger:  Ber.,  1902,  35,  4440. 

8  Bull.  107,  Bur.  Chem.,  U.  S.  Dept.  Agriculture.  For  a  review  and  discus- 
sion of  the  methods  of  determining  the  pentosans  and  the  practical  applications 
of  the  results  see  Krober,  Rimbach,  and  Tollens,  Z.  angew.  Chem.,  1902,  15, 
477,  508.  Also  Fraps  :  Am.  Chem.  J.,  1901,  25,  501, 

4  Ann.  Chem.,  1887,  243,  314. 


CARBOHYDRATES  —  GENERAL  METHODS         59 

charic,  mannosaccharic,  and  raucic  acids  being  obtained  respec- 
tively from  dextrose,  maiinose,  and  galactose. 

All  of  these  oxidation  products  are  freely  soluble  except 
mucic  acid,  which  is  practically  insoluble  in  water  or  dilute 
nitric  acid.  Since  mucic  acid  is  produced  in  fairly  constant 
proportion,  its  insolubility  affords  a  means  for  the  approximate 
determination  of  galactose  or  any  substance  which  yields  galac- 
tose on  hydrolysis. 

Mucic  Acid  Method  for   G-alactose,   Lactose,  Raffinose,  and 

Gralactans.1 

Weigh  1  to  3  grams  of  substance  according  to  the  amount  of 
mucic  acid  expected ;  remove  fat  if  necessary  by  washing  with 
ether  ;  transfer  to  a  beaker  about  5.5  cm.  in  diameter  and  T  cm. 
deep ;  add  60  cc.  of  nitric  acid  of  1.15  sp.  gr.  and  evaporate 
the  solution  to  exactly  one  third  its  volume  on  a  water  bath  at 
a  temperature  of  94°  to  96°.  After  standing  24  hours,  add  10 
cc.  of  water  to  the  precipitate  and  allow  it  to  stand  another 
24  hours.  The  mucic  acid  has  now  crystallized,  and,  unless 
contaminated  with  insoluble  residue  from  the  sample,  it  can  be 
transferred  to  a  weighed  filter,  washed  with  30  cc.  of  water,  then 
with  alcohol  and  ether,  dried  at  100°,  and  weighed. 

In  case  other  insoluble  substances  are  present,  return  the 
filter  and  mucic  acid,  after  washing  with  water,  to  the  beaker ; 
warm  15  minutes  with  a  mixture  of  1  part  strong  ammonia, 
1  part  ammonium  carbonate,  and  19  parts  of  water  :  filter  and 
wash ;  evaporate  filtrate  to  dryness  over  a  water  bath ;  add 
5  cc.  nitric  acid  of  1.15  sp.  gr.  ;  stir  thoroughly  and  allow  to 
stand  for  30  minutes.  Collect  the  mucic  acid  on  a  weighed 
filter,  wash  with  10  to  15  cc.  of  water,  then  with  60  cc.  of 
alcohol  and  a  number  of  times  with  ether ;  dry  at  100°  and 
weigh. 

When  these  directions  are  strictly  followed,  galactose  yields 
about  three  fourths  its  weight  of  mucic  acid.  The  weight  of 

1  Tollens  and  Rischbiet :  Ber.,  1885, 18,  2616.  Creydt :  Ibid.,  1886, 19,  3116. 
Bull.  107,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 


60  METHODS  OF  ORGANIC  ANALYSIS 

'mucic  acid  obtained  from  lactose,  raffinose,  or  galactan  is  calcu- 
lated as  three  fourths  the  weight  of  galactose  which  could  be 
obtained  by  hydrolysis.  The  yield  of  mucic  acid  is,  however, 
considerably  influenced  by  the  details  of  manipulation.  In 
carrying  out  the  method  comparative  determinations  with  pure 
milk  sugar  (both  alone  and  mixed  in  known  proportions  with 
the  sample  under  examination)  should  always  be  made,  as  sub- 
stances may  be  present  which  prevent  the  crystallization  of  the 
mucic  acid.1 

HYDROLYSIS  BY  DILUTE  ACIDS 

Monosaccharides,  as  the  name  implies,  cannot  be  hydrolyzed 
to  simpler  sugars.  As  a  rule,  they  are  unaffected  by  dilute 
acids  except  on  prolonged  heating,  when  they  are  gradually 
attacked,  yielding  in  part  decomposition  products  like  those 
produced  by  stronger  acids,  and  undergoing  a  partial  "  rever- 
sion "  with  the  formation  of  di-  or  polysaccharides.  Dextrose, 
for  example,  when  heated  too  long  with  dilute  hydrochloric  or 
sulphuric  acid,  is  changed  partially  to  "  isomaltose  "  and  to  dex- 
trin-like anhydrides,  such  as  "  gallisin,"  the  unfermentable  con- 
stituent of  crude  commercial  glucose.  Levulose  decomposes 
much  more  readily  than  dextrose  on  heating  with  dilute  acids. 

Disaccharides  differ  considerably  in  the  readiness  with  which 
they  are  hydrolyzed  .by  acids.  Sucrose  is  very  easily  hydro- 
lyzed, a  20  per  cent  solution  being  completely  changed  to 
invert  sugar  by  mixing  with  one  tenth  its  volume  of  concen- 
trated hydrochloric  acid  (making  about  3  per  cent  of  actual 
acid  in  the  mixture)  and  warming  to  68°  at  such  a  rate  as  to 
require  15  minutes'  heating.2 

Maltose  is  less  easily  hydrolyzed  than  sucrose,  a  2  per  cent 
solution  in  2  to  3  per  cent  hydrochloric  acid  requiring  30  to  40 

iHerzfeld:  Z.  Vereins  Rubenzucker  Ind.,  1890,  40,  265;  Abs.  Chem.  Ztg., 
1890,  14,  Rep.,  108.  Stone  and  Baird :  J.  Am.  Chem.  Soc.,  1897,  19,  119. 

2  Clerget's  method,  described  by  Wiley:  Agricultural  Analysis,  Vol.  III.,  pp. 
105-107.  According  to  Borntrager  and  to  Samelson  (Z.  angew.  Chem.,  1892, 
334  ;  1893,  690  ;  1894.  267,  351),  the  sucrose  in  such  a  mixture  is  completely 
hydrolyzed  by  standing  overnight  at  room  temperature. 


CARBOHYDRATES  —  GENERAL  METHODS         61 

minutes'  boiling,  or  2  to  3  hours'  heating  on  a  water  bath,  for 
complete  hydrolysis  to  dextrose.1  Lactose  is  also  less  readily 
hydrolyzed  than  sucrose,  the  difference  being  especially  marked 
in  the  case  of  weak  acid  or  low  temperature. 

Raffinose  is  hydrolyzed  by  dilute  mineral  acids,  slowly  in  the 
cold,  much  more  rapidly  on  boiling,  requiring  in  either  case 
more  vigorous  treatment  than  does  sucrose. 

Dextrin,  glycogen,  and  starch  are  hydrolyzed  to  dextrose 
under  the  same  conditions  as  maltose,  but  require  somewhat 
longer  heating.  Pentosans  and  galactans  (at  least  the  more 
common  forms)  are  hydrolyzed  by  acids  almost  as  readily  as 
starch.  Normal  cellulose  is  not  hydrolyzed  by  boiling  dilute 
acids.  Hemicellulose  is  a  term  commonly  applied  to  the  carbo- 
hydrate matter  in  the  cell  walls  of  plants,  more  resistant  to 
enzymes  than  starch,  but  dissolved  and  hydrolyzed  on  boiling 
with  dilute  mineral  acids.  The  hemicellulose  of  many  of  the 
common  food  plants  consists  largely  of  pentosans. 

REACTIONS  WITH   HYDRAZINES 

All  of  the  monosaccharides,  and  maltose  and  lactose  among 
the  disaccharides,  have  the  carbonyl  group  (as  such  or  in  the 
tautomeric  form),  and  therefore  on  treatment  with  hydrazines 
yield  hydrazones  or  osazones.  The  hydrazines  chiefly  used 
are  phenylhydrazine,  para-bromphenylhydrazine,  methylphe- 
nylhydrazine  (C6H5N(CH3)  •  NH2),  diphenylhydrazine,  and 
naphthylphenylhydrazine. 

Phenylhydrazine  has  been  most  commonly  employed,  and  its 
behavior  with  the  different  sugars  has  been  most  fully  investi- 
gated. The  analytical  application  of  the  phenylhydrazine  re- 
action is  discussed  below. 

The  other  hydrazines  are  used  largely  for  confirmatory  tests 
and  also  for  the  purpose  of  distinguishing  between  sugars 
whose  behavior  with  phenylhydrazine  is  not  sufficiently 

1  It  is  sometimes  stated  that  maltose  cannot  be  made  to  yield  more  than 
about  98  per  cent  of  the  theoretical  amount  of  dextrose,  the  acid  always  begin- 
ning to  attack  the  latter  before  the  hydrolysis  of  the  former  is  quite  complete. 
See  notes  on  the  determination  of  starch. 


62  METHODS  OF  ORGANIC  ANALYSIS 

different  to  permit  of  satisfactory  identification  by  that  reagent 
alone. 

Mannose  is  distinguished  from  the  other  sugars  by  forming 
•with  phenylhydrazine  an  insoluble  hydrazone,  and  this  reaction 
has  been  applied  quantitatively  by  Bourquelot  and  Herissey.1 

The  phenylhydrazones  of  the  other  sugars  are  soluble,  but 
when  heated  with  an  excess  of  phenylhydrazine  react  to  form 
osazones.2 

To  form  the  osazone,2  dissolve  one  part  of  the  sugar,  two  of 
pure  phenylhydrazine  hydrochloride,  and  three  of  crystallized 
sodium  acetate  in  20  parts  of  water  in  a  test  tube ;  filter  if  not 
clear  ;  cork'  loosely  to  avoid  evaporation  and  place  the  tube  in 
boiling  water. 

Under  these  conditions  the  maximum  yield  of  osazone  is  usu- 
ally obtained  by  warming  the  solution  in  the  water  bath  for 
one  to  two  hours  and  then  allowing  it  to  cool.  The  osazone  thus 
obtained  is  ordinarily  a  yellow  iridescent  precipitate,  more  or 
less  distinctly  crystalline  according  to  the  purity  and  concentra- 
tion of  the  solution.  To  purify  it,  filter  on  a  small  paper,  wash 
with  a  little  cold  water,  dissolve  in  the  smallest  possible  amount 
of  boiling  50  per  cent  alcohol,  and  filter  hot.  The  osazone 
which  separates  from  the  alcohol  solution  may,  if  desired,  be 
further  recrystallized  from  alcohol  or  from  pyridine. 

Dextrose  and  levulose  yield  the  same  osazone,  glucosazone, 
which  crystallizes  in  needles  melting  at  204°-205°  when  heated 
at  such  a  rate  that  the  melting  point  is  reached  in  three  to  four 
minutes.  G-alactosazone  crystallizes  in  needles  which  melt  at 
193°  ;  maltosazone,  in  independent  needles  or  tables  melting  at 
206°  ;  lactosazone,  in  masses  of  microscopic  prisms  melting  at 
200°.  Xylosazone  and  arabinosazone  melt  at  about  160°. 

A  conclusive  method  of  ascertaining  whether  an  osazone  is 
that  of  a  pentose,  a  hexose,  or  a  disaccharide  is  to  determine 
the  percentage  of  nitrogen.  In  the  case  of  a  mixture,  the  hex- 

*J.  Pharm.  Chim.,  1899,  [6],  10,  206.  See  also  Pellet :  Bui.  Assoc.  Chim. 
Sucr.  Distill,  1900-01,  18,  758  ;  Abs.  Z.  Nahr.  Genussm.,  1902,  5,  74. 

2  Fischer:  Ber  ,  1884  17,  579  ;  1887,  20,  821  ;  1888,  21,  1805,  2631;  1889, 
22,  87  ;  1890,  23,  2117. 


CARBOHYDRATES  —  GENERAL  METHODS        63 

osazone  can  be  freed  from  osazones  of  the  other  two  groups  by 
washing  with  hot  water.  Maltosazone  is  soluble  in  about  75 
parts,  xylosazone  in  about  50  parts,  and  lactosazone  in  80  to  90 
parts,  of  boiling  water,  while  the  pure  hexosazones  are  nearly 
insoluble. 

Since  the  melting  points  of  osazones  are  not  very  sharply 
defined  and  may  be  appreciably  below  the  figures  just  given 
if  the  osazones  are  not  entirely  pure  or  are  not  heated  at  a  suf- 
ficiently rapid  rate,  an  entirely  conclusive  identification  of  a 
sugar  by  means  of  phenylhydrazine  alone  is  usually  not  to  be 
expected,  but  by  similar  reactions  with  some  of  the  derivatives 
of  phenylhydrazine  above  mentioned  a  characteristic  hydrazone 
or  osazone  may  be  obtained. 

Thus  Neuberg1  identified  arabinose  by  its  diphenylhydra- 
zone  and  distinguished  between  glucose  and  fructose  by  the 
difference  in  their  behavior  toward  methylphenylhydrazine ; 
Tollens  and  Maurenbrecker 2  also  separate  arabinose  from 
xylose  by  the  preparation  of  the  diphenylhydrazone. 

Kahl3  found  that  parabrombenzylhydrazide  condenses  to 
form  an  insoluble  hydrazone  with  glucose,  galactose,  mannose, 
or  arabinose ;  not  with  levulose,  maltose,  or  lactose ;  and  to 
only  a  slight  extent  with  xylose.  Kendall  and  Sherman4 
worked  out  a  method  by  which  this  reaction  can  be  made  to 
serve  for  the  identification  of  any  one  of  the  four  reacting 
sugars,  but  the  usefulness  of  this  method  is  restricted  by  the 
fact  that  the  preparation  of  the  reagent  is  troublesome  and 
time-consuming. 

Neuberg 6  made  use  of  the  differences  in  optical  activity  of 
the  osazones  as  a  means  of  identification.  Dissolving  0.2  gram 
osazone  in  4  cc.  pyridine  plus  6  cc.  absolute  alcohol  and  examin- 
ing the  solution  in  a  100-mm.  tube  in  the  circular-scale  polari- 
scope  (see  discussion  of  polariscope  methods  beyond),  he 
obtained  the  following  results: 


1  Ber.,  33,  2243  ;  35,  959.  3  Dissertation,  Freiburg,  1904. 

2  Ber.,  38,  600.  *  J.  Am.  Chem.  Soc.,  1908,  30,  1451. 

6  Ber.,  1899,  32,  3384 


64 


METHODS   OF   ORGANIC   ANALYSIS 


Compound 

Melting  point 

Eotation 

Arabinosephenylosazone        

160° 

_|_  1°  10' 

Arabinose-p-brornphenylosazone             .          . 

196°  °00° 

-f  0°  °8' 

Xylosephenylosazone                            •                         • 

158° 

0°  15' 

Xylose-p-bromphenyloStizonc                                      • 

9Q8° 

4-0° 

204°-205° 

-  1°  30' 

222° 

—  0°  31' 

Galactosephenyloso/zone   

196°-197° 

+  0°  48' 

IVlaltosephenylosEizone            .          

206° 

-f  1°  30' 

Lactosephenylosazone      '                ...          ... 

200° 

4-  0° 

Glucuronic  acid-p-bromphenylhydrazine  compound 

216° 

-  7°  25' 

Only  rarely  are  the  rotatory  powers  of  the  osazones  of  assist- 
ance in  analytical  work,  since  the  differences  are  small  and 
difficult  to  observe. 

By  the  application  of  the  different  hydrazines  as  reagents,, 
and  the  study  of  the  products  obtained  with  reference  to  solu- 
bility, crystalline  form,  melting  point,  etc.,  it  is  possible  to 
identify  the  individual  sugars  even  in  such  mixtures  as  are  ob- 
tained by  the  hydrolysis  of  plant  tissues  or  in  complex  artificial 
mixtures  of  carbohydrates  which  do  not  occur  together  in 
nature.  For  a  full  account  of  such  methods  see  the  works  of 
Abderhalden,  Browne,  Lippmann,  Oppenheimer,  and  Tollens. 

If,  however,  the  problem  is  to  identify  a  pure  sugar  or  a 
simple  mixture  of  sugars,  it  will  usually  be  more  convenient  to 
apply  the  osazone  reaction  in  the  manner  described  below  and 
depend  upon  other  methods  to  complete  the  identification  as 
described  later. 

ANALYTICAL  APPLICATION  OF  THE  OSAZONE  REACTION 

Maqueiine1  found  that  the  reducing  sugars  when  treated  in  a 
uniform  way  with  phenylhydrazine  showed  considerable  differ- 
ences both  in  the  yield  of  osazone  and  in  the  time  required  for 
the  appearance  of  the  osazone  precipitate.  Mulliken2  has 


1  Compt.  rend.,  112,  799. 

2  Identification  of  Pure  Organic  Compounds,  Vol.  I. 


CARBOHYDRATES  —  GENERAL  METHODS         65 

studied  these  differences  in  rapidity  of  osazone  formation  and 
makes  use  of  them  to  an  important  extent  in  his  scheme,  for  the 
identification  of  pure  sugars.  According  to  Mulliken,  0.1 
gram  sugar,  0.2  gram  pure  phenylhydrazine  hydrochloride,  0.3 
gram  sodium  acetate,  and  2  cc.  water  are  mixed  in  a  small  test 
tube,  corked  loosely  to  prevent  evaporation,  and  heated  in'  boil- 
ing water.  If  the  tube  is  occasionally  shaken  without  removing 
it  from  the  boiling  water,  the  osazone  precipitate  usually  sepa- 
rates out  quite  suddenly  so  that  duplicate  experiments  usually 
give  results  that  agree  within  half  a  minute.  Under  these,  con- 
ditions the  time  required  for  the  appearance  of  the  osazone  in 
the  hot  solution  is  given  by  Mulliken  as  follows  :  Fructose,  two 
minutes  ;  sorbinose,  three  and  one  half  minutes ;  glucose,  four 
to  five  minutes ;  xylose,  seven  minutes ;  rhamnose,  nine  min- 
utes ;  arabinose,  ten  minutes ;  galactose,  fifteen  to  nineteen 
minutes.  Sucrose,  after  about  thirty  minutes'  heating,  ifs  suffi- 
ciently hydrolyzed  to  yield  a  slight  precipitate  of  glucosazone. 
Maltose  and  lactose  give  no  precipitate  in  the  hot  solution  even 
when  the  heating  is  continued  for  two  hours. 

Sherman  and  Williams1  followed  closely  the  conditions 
adopted  by  Mulliken  except  that,  for  greater  convenience  of 
manipulation,  twice  the  quantities  were  used  throughout. 
Having  confirmed  the  results  above  given  for  glucose,  fructose, 
sucrose,  maltose,  and  lactose  at  the  standard  dilution,  they  de- 
termined the  times  required  for  the  osazone  precipitation  with 
smaller  amounts  of  glucose  or  fructose  in  pure  solution  and 
also  when  different  amounts  of  other  sugars  were  present  at 
the  same  time.  Every  test  was  carried  out  as  has  been 
described!^  using  0.4  gram  phenylhydrazine  hydrochloride, 
0.6  gram  sodium  acetate,  and  4  cc.  water,2  so  that  the  only 
variable  factor  was  the  amount  of  sugar  or  sugars  present. 
The  tabular  statements  which  follow  show  the  time  of  heating 
required  for  the  appearance  of  an  osazone  precipitate  in  each 
case. 

1  J.  Am.  Chem.  Soc.,  1906,  28,  629. 

2  If  the  solution  at  this  point  is  not  clear,  it  is  filtered  through  a  dry  paper 
before  heating. 

F 


66  METHODS   OF   ORGANIC   ANALYSIS 

PURE  SOLUTIONS  OF  GLUCOSE,  FRUCTOSE,  INVERT  SUGAR,  OR  SUCROSE 


Weight  of  sugar 
taken      Gram 

Glucose 
Minutes 

Fructose 
Minutes 

Invert  sugar 
Minutes 

Sucrose 

Minutes 

0.2 

4-5 

l;l_ll 

1HI 

31 

0.1 

5 

lf-2 

2 

35 

-  0.05 

6| 

2* 

3 

78 

0.01 

17 

5£ 

6-6$ 

No  ppt. 

0.005 

34 

10 

14 

0.0025 

65 

17 

With  less  than  0.005  gram  glucose  or  0.0025  gram  fructose, 
the  amount  of  osazone  separating  in  the  hot  solution  was  small 
and  the  time  of  its  appearance  doubtful. 


INFLUENCE  OF  MALTOSE  ON  GLUCOSE 


Weight  of 
glucose 
Gram 

0.01 
0.02 

Weight  of  maltose 

0.2  grain 

No  ppt. 
26-28  min. 

0.1  gram 

40  min. 

0.05  gram 

30  rnin. 

0.01  gram 

22  min. 

Weight  of 

glucose 

Gram 

0.01 
0.02 


INFLUENCE  OF  LACTOSE  ON  GLUCOSE 

Weight  of  lactose 


0.2  gram 

No  ppt. 

0.1  gram 

50  min. 

0.05  gram 

32  min. 

0.01  gram 

25  min. 

45-48  min. 


In  absence 

of 
maltose 

17  min. 
12-13  min. 


In  absence 

of 
lactose 

17  min. 
12-13  min. 


It  is  evident  that  both  maltose  and  lactose  interfere  seriously 
with  the  formation  and  precipitation  of  glucosazone  and  that 
the  influence  of  lactose  is  greater  than  that  of  maltose.  Thus 
a  mixture  of  0.01  gram  glucose  and  0.1  gram  lactose  required 
ten  minutes'  longer  heating  than  a  parallel  mixture  with  0.1 
gram  maltose,  and  when  the  quantities  of  glucose,  lactose,  and 
maltose  are  doubled  (the  amounts  of  reagents  and  the  volume  of 
the  solution  remaining  the  same)  the  lactose  mixture  required 
twenty  minutes'  longer  heating  than  the  maltose  mixture. 

INFLUENCE  OF  SUCROSE  ON  GLUCOSE 


Weight  of 
glucose 
Gram 

0.005 
0.01 

AVeight  of  sucrose 

In  absence 
of 

sucrose 

33-39  min. 
17  min. 

0.2  gram 

15-17  min. 
14-16  min. 

0.1  gram 

15-17  min. 
16  min. 

0.05  gram 

22  min. 
17  min. 

0.01  gram 

30  min. 
17  min. 

CARBOHYDRATES  —  GENERAL  METHODS 


67 


Weight  of 

fructose 

Gram 

0.01 


Weight  of 

fructose 

Gram 

0.01 


INFLUENCE  OF  MALTOSE  ON  FRUCTOSE 

Weight  of  maltose 


0.2  gram 

7-8  min. 


0.1  gram 

min. 


0.05  gram 


0.01  gram 

5|  min. 


INFLUENCE  OF  LACTOSE  ON  FRUCTOSE 

Weight  of  lactose 


0.2  gram 

MO  min. 


0.1  gram 

7|  min. 


0.05  gram 

6|  min. 


0.01  gram 

6  min. 


In  absence 

of 
maltose 

5    min. 


In  absence 

of 
lactose 

5    min. 


Comparing  these  results  with  the  corresponding  figures  for 
glucose,  it  will  be  seen  that  the  interference  of  maltose  and  lac- 
tose is  less  marked  with  fructose  than  with  glucose.  In  both 
cases,  however,  the  appearance  of  the  osazone  precipitate  is  re- 
tarded distinctly  by  maltose  and  to  a  greater  extent  by  lactose. 


Weight  of 

fructose 

Gram 

0.005 


INFLUENCE  OF  SUCROSE  ON  FRUCTOSE 

Weight  of  sucrose 


0.2  gram 

8    min. 


0.1  gram 

8|  min. 


0.05  gram 

9i  min. 


0.01  gram 

9    min. 


In  absence 

of 
sucrose 

9i  min. 


Here  the  effect  of  the  sucrose  was  not  so  noticeable. 

Interpretation  of  time  relations  in  applying  the  osazone  test  to 
sugar  mixtures  or  to  samples  in  which  the  concentration  of  the 
reacting  sugar  is  not  known  or  is  different  from  that  of  the 
conventional  method,  should  be  based  on  a  careful  study  of 
the  above  data. 

The  delicacy  of  the  test  in  the  presence  of  dextrin  (which  is 
known  to  retard  the  formation  of  the  osazone),  and  of  sub- 
stances other  than  carbohydrates,  has  not  been  studied  in  de- 
tail, except  in  the  case  of  certain  constituents  of  urine.1  Hence 
in  applying  the  osazone  test  to  an  unknown  solution,  it  is  well 
to  make  at  the  same  time  two  check  experiments,  one  with  a 
mixture  of  carbohydrates  corresponding  to  that  which  the  un- 
known solution  is  believed  to  contain,  the  other  with  a  portion 

1  The  influence  of  other  substances  likely  to  be  present,  upon  the  osazone  test 
for  dextrose  in  urine  has  been  studied  by  Hirschl :  Z.  physiol.  Chem.,  1890,  14, 
377.  Jaff£:  Ibid.,  22,  532.  Neuberg:  Ibid.,  1900,  20,  274.  Neumann- Wender : 
Pharmac.  Post.,  26,  673,  614  (Vaubel  ;  I.  c.,  II, 


68  METHODS  OF  ORGANIC  ANALYSIS 

of  the  unknown  solution  to  which  has  been  added  a  very  small 
amount  of  dextrose. 

The  yield  of  osazone  has  been  studied  especially  by  Maquenne,1 
Laves,2  Fischer,3  Lintrier  and  Krober,4  Raimann,5  and  Davis 
and  Ling.6  In  each  of  these  cases  the  osazone  was  formed  by 
heating  for.  one  or  two  hours,  in  a  more  dilute  solution  than 
that  above  given,  with  a  considerable  excess  of  a  slightly  acid 
solution  of  phenylhydrazine  acetate.  The  precipitate  of  osa- 
zone can  then  be  filtered,  washed  with  water,  and  weighed. 
According  to  Laves  the  following  corrections  should  be  applied 
for  the  amount  of  glucosazone  left  in  solution  or  dissolved  by 
washing  : 

100  parts  boiling  water  dissolve 0.01      part  osazone 

100  parts  water  at  20°  dissolve        0.0042  part  osazone 

100  parts  2  per  cent  acetic  acid  at  20°  dissolve  .  .  .  0.007  part  osazone 
100  parts  3  per  cent  acetic  acid  at  20°  dissolve  .  .  .  0.0145  part  osazone 
100  parts  4  per  cent  acetic  acid  at  20°  dissolve  .  .  .  0.022  part  osazone 
100  parts  5  per  cent  acetic  acid  at  20°  dissolve  .  .  .  0.031  part  osazone 
100  parts  10  per  cent  alcohol  slightly  acidulated  dis- 
solve at  20°  0.0075  part  osazone 

Corresponding  corrections  have  not  been  worked  out  for 
other  osazones,  as  the  attempts  to  apply  the  osazone  method 
quantitatively  have  been  mainly  with  the  object  of  determining 
dextrose  or  dextrose  and  levulose.  In  comparative  tests  carried 
out  in  exactly  the  same  manner,  the  weight  of  osazone  obtained 
is  proportional  to  the  amount  of  sugar  originally  present,  but 
slight  differences  of  manipulation  affect  the  yield  to  such  an 
extent  that  the  osazone  precipitation  cannot  yet  be  regarded  as 
a  satisfactory  quantitative  method. 

Williams  7  has  recently  studied  the  conditions  affecting  the 
yield  of  glucosazone  from  pure  glucose  solutions.  It  was  found 
best  to  allow  one  hour  of  heating  for  the  formation  of  the 

1  Compt.  rend.,  1891,  112,  799 ;  Z.  anal.  Chem.,  1894,  33,  226. 

2  Archiv.  der  Pharm.,  1893,  231,  366.     Vaubel's  Bestimmung  organischer 
Verbindungen,  II,  307,  311,  312.  3  Ber.,  1895,  28,  1437. 

4  Z.  /.  d.  ges.  Brauwesen,  1895,  18,  153;  Z.  anal.  Chem.,  1896,  35,  95; 
Analyst,  1897,  20,  167.  5  Z.  anal.  Chem.,  1901,  40,  390. 

6  J.  Chem.  Soc.,  1904,  85,  24.  7  Data  not  yet  published. 


CARBOHYDRATES  —  GENERAL  METHODS         69 

osazone,  since  longer  heating,  although  increasing  the  yield, 
gave  a  less  pure  product.  A  solution  of  phenylhydrazine 
acetate  made  by  dissolving  the  free  base  in  acetic  acid  gave  as 
good  results  as  the  reagent  made  by  mixing  phenylhydrazine 
hydrochloride  with  sodium  acetate  and  was  found  more  conven- 
ient. The  amounts  of  phenylhydrazine  and  acetic  acid  greatly 
influenced  the  yield  of  osazone.  In  a  series  of  experiments  in 
which  0.2  gram  pure  dextrose  in  20  cc.  water  was  heated  with 
varying  amounts  of  reagents  for  one  hour,  then  washed  with 
100  cc.  water,  dried  and  weighed,  it  was  found  that  maximum 
yields,  about  60  per  cent  of  the  theoretical,  were  obtained  by 
using  2  grams  of  phenylhydrazine  in  8  cc.  of  50  per  cent  acetic 
acid,  2.4  grams  phenylhydrazine  in  9.2  cc.  of  50  per  cent  acetic 
acid,  or  3.2  grams  of  phenylhydrazine  in  6.4  cc.  of  50  per  cent 
acetic  acid.  Thus  while  a  large  excess  of  phenylhydrazine  was 
always  essential  to  high  yields,  the  amount  required  for  a  maxi- 
mum yield  depended  upon  the  ratio  between  the  phenylhydra- 
zine and  the  acetic  acid,  as  well  as  upon  that  between  the 
reagents  and  the  sugar. 

REDUCTION  OF   COPPER  SOLUTIONS 

The  same  sugars  which  react  with  phenylhydrazine  have  the 
power  of  reducing  certain  metallic  salts,  especially  salts  of 
copper,  silver,  and  mercury  in  alkaline  solution.  This  "re- 
ducing power,"  due  to  the  susceptibility  of  the  carbonyl  sugars 
to  oxidation,  can  be  utilized  for  the  detection  and  determina- 
tion of  these  sugars. 

When  for  example  an  alkaline  cupric  solution,  such  as-  Feh- 
ling's,  is  boiled  with  one  of  these  "  reducing "  sugars,  the 
copper  is  reduced  to  the  cuprous  state  and  the  sugar  is  at- 
tacked in  two  ways :  (1)  oxidation  and  (2)  decomposition  by 
the  alkali.  Tartronic  acid  is  often  cited  as  a  typical  oxida- 
tion product  formed  by  the  action  of  boiling  alkaline  copper 
solution  upon  dextrose,  but  in  practice  less  than  half  as  much 
copper  is  reduced  as  would  correspond  to  a  complete  oxidation 
of  glucose  to  tartronic  acid,  because  the  boiling  alkali  so  largely 
decomposes  the  glucose  into  other  than  oxidation  products. 


70  METHODS    OF   ORGANIC    ANALYSIS 

In  order  that  the  amount  of  copper  reduced  may  indicate 
quantitatively  the  amount  of  reducing  sugar,  it  is  necessary 
to  control  the  decomposing  action  of  the  alkali.  This  is  usu- 
ally accomplished  by  prescribing  the  amount  and  final  dilu- 
tion of  the  alkaline  solution  and  either  (1)  adjusting  the  sugar 
solution  so  as  to  find  the  amount  which  in  measured  volume 
will  exactly  reduce  the  copper  as  described  below  for  Fehling's 
volumetric  method,  or  (2)  treating  a  fixed  amount  of  the 
reagent  with  a  prescribed  volume  of  the  reducing  sugar  solu- 
tion of  a  strength  insufficient  to  reduce  all  the  copper  and 
collecting  and  weighing  the  copper  reduced  as  in  Defren's 
gravimetric  method  described  later. 

FEHLING'S  VOLUMETRIC  METHOD 

Reagents.  —  (1)  Copper  solution  :  Dissolve  34.64  grams  of 
pure  crystallized  copper  sulphate  in  water,  and  dilute  to  ex- 
actly 500  c.c.  • 

(2)  Alkaline  tartrate  solution :  Dissolve  175  grams  of  pure 
sodium  potassium  tartrate  and  50  grams  of  pure  sodium  hy- 
droxide in  water,  and  dilute  to  500  c.c.  This  reagent  does  not 
keep  well  unless  carefully  protected  from  the  air  (Note  1). 

Determination.  —  Measure  accurately  into  a  small  flask  or 
casserole  or  a  deep  porcelain  dish,  5  cc.  of  each  of  the  above 
solutions,  making  10  cc.  of  the  "mixed  Fehling  reagent." 
Add  40  cc.  of  water,  mix,  and  boil.  To  the  boiling  liquid, 
add  from  a  burette  a  solution  which  contains  not  over  1  per 
cent  of  the  reducing  sugar  to  be  determined,  boiling  two 
minutes  after  each  addition  of  sugar  until  the  blue  color  is 
entirely  discharged,  showing  that  all  of  the  copper  has  been 
reduced.  This  test  indicates  approximately  the  amount  of 
reducing  sugar  in  the  sample.  Now  adjust  the  strength  of  the 
sugar  solution,  if  necessary,  so  that  about  20  cc.  will  be  re- 
quired to  reduce  10  cc.  of  the  mixed  Fehling  reagent.  Repeat 
the  test,  adding  the  calculated  amount  of  sugar  solution  at 
once  to  the  boiling  copper  solution ;  regulate  the  heating  so 
that  the  mixture  will  again  begin  to  boil  about  one  minute 


CARBOHYDRATES  —  GENERAL  METHODS         71 

after  the  addition  of  the  sugar ;  note  the  exact  time  that  actual 
boiling  commences  and  continue  to  boil  for  just  two  minutes, 
then  remove  the  flame  and  at  once  test  the  liquid  for  unre- 
duced copper  (Note  2). 

Repeat  the  test,  using  more  or  less  of  the  sugar  solution 
depending  upon  the  presence  or  absence  of  an  excess  of  copper 
in  the  preceding  experiment  until  two  amounts  of  sugar  solu- 
tion are  found  which  differ  by  only  0.1  or  0.2  cc.,  one  giving 
complete  reduction  and  the  other  leaving  a  small  amount  of 
copper  in  the  cupric  state.  The  mean  of  these  two  amounts 
is  taken  as  the  volume  of  solution  required  for  complete  reduc- 
tion of  the  copper  reagent. 

Note  1. — All  of  the  reagents  used  must  be  the  purest  ob- 
tainable, and  the  two  constituents  of  the  "mixed  Fehling 
reagent "  must  be  kept  separate  instead  of  being  made  up  in 
one  solution  as  was  formerly  done.  If  the  solutions  are  not 
fresh,  make  a  blank  test  in  a  casserole,  boiling  as  above  with- 
out the  addition  of  any  sugar,  allow  to  stand  for  a  few 
minutes,  then  decant  off  the  liquid  and  notice  whether  any 
cuprous  oxide  has  been  precipitated.  Finally  wipe  out  the 
casserole  with  a  small  piece  of  filter  paper  and  examine  the 
latter,  which  may  show  traces  of  cuprous  oxide  not  visible  in 
the  presence  of  the  blue  solution.  If  the  liquid  shows  any 
change  of  color  in  this  blank  test,  or  if  the  slightest  trace  of 
cuprous  oxide  is  found,  the  alkaline  tartrate  solution  must  be 
rejected  and  another  blank  test  made  with  a  freshly  prepared 
solution. 

Note  2.  —  So  long  as  the  solution  shows  a  distinct  blue  color 
it  is  unnecessary  to  apply  other  tests  for  cupric  copper. 
When  the  test  is  made  in  a  flask,  the  blue  color  is  best  seen  by 
holding  level  with  the  eye  and  looking  horizontally  through 
the  meniscus,  but  on  account  of  the  presence  of  the  cuprous 
oxide  the  disappearance  of  the  blue  color  is  not  alone  a  safe 
criterion.  When  the  blue  color  is  no  longer  apparent,  test  for 
cupric  copper  by  one  of  the  following  methods.  It  is  well  for 
each  analyst  to  try  all  three  methods  and  select  the  one  which 
he  finds  most  satisfactory  for  the  conditions  of  his  work. 


72  METHODS   OF   ORGANIC   ANALYSIS 

Whichever  test  is  used  it  is  important  to  work  quickly  lest 
reduced  copper  be  oxidized  by  contact  with  the  air. 

Ferrocyanide  Test.  —  Quickly  filter  a  portion  of  the  liquid 
through  two  or  three  thickness  of  paper,  repeating  the  filtration 
if  necessary,  observe  carefully  that  the  filtrate  is  free  from  any 
trace  of  cuprous  oxide,  then  acidulate  with  acetic  acid  and  add 
a  few  drops  of  a  dilute  solution  of  potassium  f errocyanide  when 
if  copper  is  present  a  red-brown  coloration  or  precipitate  will 
appear. 

Watts  and  Tempany  suggest  as  a  modification  of  this  test 
that  the  liquid  instead  of  filtering  be  poured  upon  a  small  pad 
consisting  of  several  layers  of  filter  paper,  and  the  bottom  paper 
(which  should  be  wet  with  the  solution  but  free  from  cuprous 
oxide)  be  removed,  acidulated  with  acetic  acid,  and  then  tested 
with  ferrocyanide. 

Starch-iodide  Test.1  —  As  soon  as  the  sugar  solution  and  the 
copper  reagent  have  been  boiled  together  for  the  required  two 
minutes,  add  a  drop  or  two  of  the  solution  (which  need  not  be 
filtered  from  cuprous  oxide)  to  a  considerable  excess  of  cold 
acidulated  starch-iodide  solution,  when,  if  cupric  copper  is 
present,  a  blue  or  purple  coloration  is  obtained. 

The  starch-iodide  solution  is  prepared  as  follows:  Boil  0.02 
gram  starch  with  15  to  20  cc.  of  water,  cool,  add  4  to  5  grams 
of  potassium  iodide  and  dilute  to  50  cc.  A  fresh  solution  must 
be  prepared  each  day.  When  the  test  is  to  be  made,  pour  about 
1  cc.  of  this  starch-iodide  solution  into  a  test  tube,  add  two  or 
three  drops  of  acetic  acid  and  then  immediately  a  drop  or  two 
of  the  solution  to  be  tested. 

Thiocyanate  Test.2  —  This  requires  a  solution  of  ferrous  thio- 
cyanate  which  may  be  prepared  as  follows  :  Dissolve  1  gram 
ferrous  sulphate  and  1.5  gram  ammonium  thiocyanate  in  10  cc. 
water  at  about  45°  to  50°  C.,  cool  immediately,  add  2|  cc.  con- 
centrated hydrochloric  acid  and  a  trace  of  zinc  dust  to  decolor- 
ize the  solution. 

1  Harrison:  Pharm.  J.,  1903,  170  (Button's  Volumetric  Analysis,  9th  Ed., 
p.  312.) 

2  Ling  and  Rendle  :  Analyst,  1905,  30,  183.     Ling  and  Jones  :    IUd.,  1908, 
33,  160. 


CARBOHYDRATES  —  GENERAL  METHODS         73 

Place  a  drop  of  the  freshly  prepared  ferrous  thiocyanate 
solution  on  a  porcelain  plate  and  add  a  drop  of  the  solution  to 
be  tested  (which  need  not  be  filtered),  when,  if  cupric  copper  is 
present,  the  well-known  red  color  of  ferric  thiocyanate  will  be 
produced. 

Calculation  and  Verification  of  Results.  —  In  calculating  the 
results  it  is  commonly  assumed  that  10  cc.  of  the  mixed  Fehl- 
ing  reagent  require  for  reduction  under  the  above  conditions: 

0.0500  gram  of  anhydrous  dextrose,  levulose,  or  invert  sugar. 
0.0678  gram  of  dry  crystallized  lactose  (C12H22On  -  H2O). 
0.0807  gram  of  anhydrous  maltose. 

That  these  factors  are  not  absolute,  but  are  dependent  upon 
exact  uniformity  of  conditions  has  already  been  explained.  To 
secure  the  greatest  accuracy,  therefore,  the  result  obtained 
should  be  verified  by  a  check  experiment,  carried  out  under  the 
exact  conditions  of  the  analysis,  with  a  known  solution  of  pure 
sugar  of  the  kind  actually  determined. 

It  is  sometimes  convenient  to  express  the  reducing  power  of 
some  other  sugar  or  of  a  mixture  in  terms  of  the  reducing 
power  of  dextrose,  the  latter  being  taken  as  100. 

Thus,  if  0.0678  gram  lactose  or  0.0807  gram  maltose  has  the 
same  reducing  power  as  0.05  gram  dextrose,  then  on  the  basis 

of  dextrose  =  100,  the  reducing  power  of  lactose  is  — X  100 

05 

=  74,  and  that  of  maltose  is  — X  100  =  62. 

.0807 

According  to  Soxhlet,  levulose  and  galactose  have  distinctly 
less  reducing  power  than  dextrose.  It  is  quite  commonly  as- 
sumed, however,  that  these  three  monosaccharides  have  the 
same  reducing  power,  but  that  invert  sugar  often  fails  to  show 
its  full  effect  because  of  the  decomposing  action  of  the  acid  used 
for  inversion.  Xylose  and  arabinose  reduce  Fehling's  solution 
somewhat  more  strongly  than  does  dextrose. 


74  METHODS   OF   ORGANIC   ANALYSIS 

DEFREN'S  GRAVIMETRIC  METHOD1 

This  method  is  based  on  that  of  O'Sullivan2  and  provides  a 
uniform  procedure  for  the  determination  of  dextrose,  maltose, 
or  lactose. 

Reagents.  —  (1)  Dissolve  34.64  grams  of  copper  sulphate  in 
water,  add  0.5  cc.  strong  sulphuric  acid,  and  dilute  to  500  cc. 

(2)  Dissolve  178  grams  of  sodium  potassium  tartrate  and  50 
grains  of  sodium  hydroxide  in  water  and  dilute  to  500  cc. 

Determination.  —  Mix  15  cc.  of  each  of  the  above  reagents  in 
an  Erlenmeyer  flask  having  a  capacity  of  250  to  300  cc.,  dilute 
with  50  cc.  of  freshly  boiled  distilled  water,  and  place  the  flask 
in  a  boiling  water  bath  for  five  minutes ;  then  add  25  cc.,  ac- 
curately measured  from  a  burette  or  pipette,  of  a  solution  con- 
taining approximately  0.5  per  cent  of  the  sugar  to  be  deter- 
mined and  allow  the  mixture  to  stand  in  the  boiling  water  bath 
for  fifteen  minutes.  Remove  the  flask  from  the  bath  and  filter 
at  once  (using  moderate  suction)  through  asbestos  prepared  as 
described  below  (Note  1);  wash  the  cuprous  oxide  with  boiling 
distilled  water  until  the  filtrate  is  no  longer  alkaline.  The  cu- 
prous oxide  can  now  be  (1)  washed  with  alcohol  and  then  with 
ether,  dried  in  a  boiling  water  oven  for  20  minutes,  and  weighed 
(Note  2) ;  (2)  ignited  and  weighed  as  cupric  oxide,  as  recom- 
mended by  Defren  ;  or  (3)  dissolved  in  nitric  acid  and  the  copper 
determined  by  electrolysis  or  by  any  other  reliable  method,  in 
which  case  it  will  not  be  necessary  to  use  asbestos  especially  pre- 
pared by  boiling  with  acid  and  alkali.  From  the  weight  of  copper 
or  cuprous  oxide  determined,  calculate  the  equivalent  amount 
of  cupric  oxide  and  find  the  corresponding  weight  of  reducing 
sugar  from  Defren's  table  (Note  3). 

Note  1.  —  The  filtrate  from  the  cuprous  oxide  must  always 
be  distinctly  blue,  showing  that  a  sufficient  excess  of  Fehling 
solution  was  used,  otherwise  the  determination  must  be  repeated, 
using  a  more  dilute  solution  of  the  reducing  sugar.  If  the 
copper  reduced  is  to  be  weighed  as  cuprous  or  cupric  oxide  on 
the  asbestos  filter,  the  latter  must  be  especially  prepared  in 

*J.  Amer.  Chem.  Soc.,  1396,  18,  749.  2J.  Chem.  Soc.,  1876,  30,  130. 


CARBOHYDRATES  —  GENERAL  METHODS         75 

order  that  it  shall  lose  no  weight  when  treated  with  the  hot 
alkaline  Fehling  solution.  Asbestos  of  good  quality  is  boiled 
with  nitric  acid  (1.05  to  1.10  sp.  gr.),  washed  with  water,  then 
boiled  with  25  per  cent  sodium  hydroxide,  washed,  and  the 
treatment  with  acid  and  alkali  repeated.  The  prepared  asbes- 
tos is  used  to  make  a  tight  felt  about  1  centimeter  thick  in  a 
Gooch  crucible.  When  the  crucible  has  been  prepared  for  use 
and  weighed,  it  should  be  tested  by  running  through  it  a 
"  blank "  of  hot  alkaline  Fehling  solution  and  washing  with 
water  as  in  a  regular  determination.  The  loss  of  weight  should 
not  exceed  one  half  milligram.  After  each  determination  the 
precipitate  is  dissolved  in  nitric  acid,  and  the  crucible  washed, 
ignited,  and  reweighed.  If  a  loss  of  over  one  milligram  is 
found,  the  determination  should  be  rejected  and  the  filter 
treated  alternately  with  acid  and  alkali  until  it  ceases  to  lose  in 
weight. 

Note  2.  — The  method  of  weighing  the  cuprous  oxide  as  such 
is  convenient,  and  when  working  with  fairly  pure  sugar  mixtures 
is  accurate,  but  impure  material,  such  as  crude  raw  sugar, 
molasses,  or  malt  extract,  is  liable  to  leave  some  organic  matter 
with  the  cuprous  oxide  on  the  filter  so  that  for  the  most  accurate 
results  with  such  materials,  one  should  ignite  to  cupric  oxide  or 
dissolve  and  determine  copper. 

Note  3.  —  Defren  determined  the  amount  of  copper  reduced 
by  fifteen  to  twenty  known  solutions  each  of  dextrose,  maltose, 
and  lactose,  with  the  following  results: 

Dextrose=  (0.4400  +  0.000037  W)  W. 
Maltose  =(0.7215  +  0.000061  W)W. 
Lactose  =(0.6270  +  0.000053  W)W. 

In  which  Wis  the  weight  of  cupric  oxide  obtained,  the  values 
of  W  varying  from  30  to  320  milligrams. 

From  these  formulae  it  is  apparent  that  the  reducing  power 
of  each  of  the  sugars  increases  slightly  with  the  concentration  ; 
hence,  to  find  the  amount  of  reducing  sugar  corresponding  to 
any  given  weight  of  copper,  one  must  use,  not  a  simple  factor, 
but  a  formula  or  (more  conveniently)  a  table. 


76 


METHODS  OF  ORGANIC  ANALYSIS 


Table  7  is  condensed  from  that  given  in  Defren's  paper  (Z.  <?.), 
in  which  the  weights  of  sugars  corresponding  to  each  milligram 
of  cupric  oxide  are  stated.  In  using  either  table  the  weight  of 
copper  or  cupric  oxide  should  be  taken  to  one  tenth  milligram 
and  the  corresponding  weight  of  reducing  sugar  found  by  the 
method  of  proportional  parts. 

TABLE?.  —  DEFREN'S  TABLE  FOR  DEXTROSE,  MALTOSE,  AND  LACTOSE 


Cupric 
Oxide 
Mgms. 

Dextrose 
Mgms. 

Maltose 
Mgms. 

Lactose 
Mgms. 

Cupric 
Oxide 
Mgtns. 

Dextrose 
Mgms. 

Maltose 
Mgms. 

Lactose 
Mgms. 

30 

13.2 

21.7 

18.8 

180 

80.4 

131.8 

114.6 

40 

17.6 

29.0 

25.2 

190 

84.9 

139.1 

121.0 

50 

22.1 

36.2 

31.5 

200 

89.5 

146.6 

127.5 

60 

26.5 

43.5 

37.8 

210 

94.0 

.154.1 

134.1 

70 

30.9 

50.8 

44.1 

220 

98.6 

161.5 

140.6 

80 

35.4 

58.1 

50.5 

230 

103.2 

169.1 

147.0 

90 

39.9 

65.5 

56.8 

240 

107.7 

176.6 

153.5 

100 

44.4 

72.8 

63.2 

250 

112.3 

184.1 

160.0 

110 

48.9 

80.1 

69.5 

260 

116.9 

191.6 

166.5 

120 

53.3 

87.4 

75.9 

270 

121.4 

199.2 

173.0 

130 

57.8 

94.8 

82.4 

280 

126.1 

206.8 

179.6 

140 

62.2 

102.1 

88.7 

290 

130.7 

214.3 

186.2 

150 

66.8 

109.5 

95.2 

300 

135.3 

221.9 

192.8 

160 

71.3 

116.9 

101.7 

310 

139.9 

229.6 

199.3 

170 

75.8 

124.4 

108.2 

320 

144.5 

237.2 

205.9 

On  account  of  the  difference  in  experimental  conditions  the 
relative  reducing  powers  of  dextrose,  maltose,  and  lactose  do  not 
show  exactly  the  same  ratios  for  this  method  as  for  the  volu- 
metric method  described  above. 


BARFOED'S  CUPRIC  ACETATE  METHOD1 

For  distinguishing  dextrose  from  maltose  Barfoed  used  a 
slightly  acid  solution  of  cupric  acetate,  which  is  reduced  by  the 

1  Barfoed  :  Z.  anal.  Chem.,  1873,  12,27.  Miiller  and  Hagen  :  Arch.  ges. 
Physiol.  (Pfluger),  22,  325.  Lieben  :  Z.  Vereins  Bub e mucker- Industrie,  1884, 
34,  857  ;  Wiley,  Agricultural  Analysis,  Vol.  Ill,  p.  291. 


CARBOHYDRATES  —  GENERAL  METHODS         77 

former  and  not  by  the  latter.  Lieben  attempted  to  determine  dex- 
trose quantitatively  in  the  presence  of  maltose  by  means  of  such 
solutions.  This  test,  like  the  formation  of  the  osazone,  is  not 
of  much  quantitative  value  unless  for  comparisons  under  identi- 
cal conditions,  but  is  often  useful  as  a  qualitative  method  for 
distinguishing  between  dextrose  (or  other  monosaccharide)  and 
those  disaccharides  which  also  reduce  Fehling's  solution. 

Reagent.  —  Dissolve  45  grams  of  neutral  crystallized  cupric 
acetate  in  900  cc.  of  water,  filter  if  necessary;  add  1.2  cc.  of 
50  per  cent  acetic  acid  and  dilute  to  a  liter.  A  portion  of  this 
reagent  heated  in  a  water  bath  must  show  no  change. 

Test.  —  To  5  cc.  of  this  reagent  in  a  test  tube  add  5  cc.  of  the 
solution  to  be  tested  and  place  in  a  boiling  water  bath  for  3J 
minutes  ;  examine  for  cuprous  oxide,  viewing  the  tube  against 
a  black  background  in  a  good  light.  If  no  evidence  of  reduction 
is  found,  allow  the  tube  to  stand  at  room  temperature  for  5  or 
10  minutes  and  examine  again.  For  uniform  results  the  differ- 
ent tubes  used  in  making  the  tests  should  be  of  nearly  the  same 
diameter  and  thickness  of  wall.  It  is  sometimes  advantageous 
to  pour  out  the  liquid  at  the  end  x)f  the  test,  leaving  any  cuprous 
oxide  as  far  as  possible  adhering  to  the  bottom  of  the  test  tube, 
and  examine  again. 

It  has  been  found l  that  the  test  .when  carefully  carried  out 
in  this  manner  was  efficient  for  the  detection  of  0.0004  gram 
of  glucose,  either  alone  or  in  the  presence  of  maltose,  lactose,  or 
sucrose  up  to  0.02  gram. 

Reduction  due  to  disaccharide  occurs  if  too  much  either  of 
sugar  or  of  acid  be  present,  or  if  the  heating  be  too  prolonged. 

In  order  to  effect  complete  destruction  of  the  glucose,  so  that 
the  filtrate  might  be  utilized  in  testing  for  maltose  or  lactose,  it 
was  necessary  to  limit  the  amount  to  about  0.002  gram  of  glucose 
to  5  cc.  of  the  reagent. 

The  test  requires  very  careful  regulation  as  to  details  of  ma- 
nipulation and  amount  of  sugar  tested,  but  under  such  restric- 
tions is  capable  of  greater  usefulness  than  has  generally  been 
appreciated. 

1  Hinkel  and  Sherman :  J.  Am.  Chem.  Soc.,  29,  1744. 


78  METHODS   OF   ORGANIC   ANALYSIS 

On  account  of  the  difficulty  of  securing  an  exact  degree  of 
acidity  in  the  cupric  acetate  solution,  each  chemist  should 
demonstrate  the  efficiency  of  his  reagent,  as  well  as  verify  his 
manipulation,  by  check  experiments  upon  known  sugar  solutions 
covering  the  probable  range  of  composition  of  the  unknown 
solutions  to  be  tested. 

OTHER  COPPER  REDUCTION  METHODS 

Until  recently  the  methods  based  upon  copper  reduction  were 
for  the  most  part  worked  out  for  the  different  reducing  sugars 
individually,  with  the  result  that  the  methods  and  tables  were 
applicable  only  to  individual  sugars.  The  method  of  Defren 
unifies  the  determination  of  dextrose,  lactose,  and  maltose. 
The  table  of  Allihn  given  beyond  (Chapter  V),  while  devised 
only  for  dextrose,  has  been  so  widely  used  that  some  workers 
have  preferred  to  use  the  same  procedure  and  table  for  other 
reducing  sugars  with  a  factor  to  convert  the  results  from  ap- 
parent glucose  to  the  sugar  sought.  The  destruction  of  reduc- 
ing sugar  by  the  decomposing  action  of  the  alkali  noted  above 
as  a  source  of  error  in  working  with  the  ordinary  Fehling  solu- 
tion, is  still  greater  in  Allihn's  method,  which  employs  a  more 
strongly  alkaline  solution.  To  avoid  this  the  caustic  alkali  may 
be  replaced  by  a  weaker  alkali  such  as  carbonate  (Soldani, 
Sidersky,  Ost,  Benedict)  or  ammonia  (Allein  and  Gaud). 
Others  have  advocated  the  addition  of  ammonia  (Pavy)  or 
cyanide  (Gerard)  to  the  usual  Fehling  solution  in  order  to 
hold  in  solution  the  reduced  copper  so  that  the  point  of  com- 
plete reduction  may  be  directly  observed  by  the  disappearance 
of  the  blue  color. 

These  methods  possess  obvious  points  of  advantage;  but  as 
yet  have  not  come  into  such  general  use  as  the  Fehling,  Allihn, 
and  Defren  methods. 

ROTATION  OF  POLARIZED  LIGHT 

Solutions  of  the  natural  carbohydrates  have  the  property 
of  rotating  the  plane  of  polarized  light.  This  rotating  power 


CARBOHYDRATES  —  GENERAL  METHODS         79 

is  also  shown  by  most  other  natural  substances  containing  one 
or  more  asymmetric  carbon  atoms  in  the  molecule. 

PREPARATION  OF  SOLUTIONS  FOR  POLARIZATION 

Solutions  for  polarization  must  be  clear  and  free  from  all 
impurities  having  an  influence  upon  polarized  light.  Among 
the  optically  active  substances  most  likely  to  be  met  in  natural 
products  are  organic  acids  (especially  tartaric),  pectin  bodies, 
gums,  resins,  coloring  matters,  glucosides,  alkaloids,  and  proteins. 
In  most  cases  the  interfering  substances  can  be  precipitated 
and  the  solution  clarified  by  adding,  first,  basic  lead  acetate, 
and  then  a  cream  of  aluminium  hydroxide.  It  is  sometimes 
advantageous  to  add  a  small  amount  of  tannin  before  clarifying. 
Tannin  combines  with  many  of  the  substances  mentioned, 
either  precipitating  them  or  forming  compounds  more  easily 
precipitated  by  the  basic  acetate.  In  case  the  solution,  after 
being  treated  with  clarifying  agents,  made  up  to  definite 
volume  and  filtered,  is  too  highly  colored  for  examination  in 
the  polariscope,  it  may  be  poured  slowly  through  a  small 
amount  of  pure  dry  animal  charcoal  on  a  paper  filter.  Since 
the  charcoal  absorbs  a  small  amount  of  the  sugar,  the  first  por- 
tions of  the  filtrate  must  be  rejected.  The  technical  details  of 
clarifying  sugar  solutions  for  polarization  will  be  discussed  in 
the  next  chapter.  A  solution  of  pure  sugar  or  mixture  of  pure 
sugars  should  not  require  the  use  of  clarifying  agents,  but 
should  be  filtered  to  remove  dust  particles. 

Reducing  sugars  when  first  dissolved  often  show  multirota- 
tion  due  to  the  spacial  arrangement  of  the  tautomeric  form  of 
the  carbonyl  group,  for  explanation  of  which  reference  may  be 
made  to  the  latest  edition  of  Hollemann's  Organic  Chemistry, 
or  to  Armstrong's  Simple  Carbohydrates  and  Glucosides.  The 
"  normal "  rotation  is  established  by  allowing  the  solution  to 
stand  for  some  hours,  by  boiling,  or  by  the  addition  of  about 
0.1  per  cent  of  ammonia.  Solutions  containing  ammonia 
darken  on  standing  and  should  therefore  be  polarized  as  soon 
as  prepared.  Sucrose  does  not  show  multirotation. 


80 


METHODS  OF  ORGANIC  ANALYSIS 


DETERMINATION  OF  ANGULAR  ROTATION 

The  standard  instrument  for 
the  direct  measurement  of  angu- 
lar rotation  is  the  Laurent  polari- 
scope  or  one  of  its  modifications. 
This  has  a  circular  scale  gradu- 
ated in  angular  degrees.  The 
principal  parts  of  an  instrument 
of  this  type  are  indicated  in  the 
diagram,  Fig.  6.  With  this  po- 
lariscope  the  rotation  produced 
by  the  active  substance  is  found 
by  turning  the  analyzing  prism, 
which  is  fixed  in  position  in  re- 
gard to  the  scale  but  rotates  on 
its  axis.  The  zero  point  is  found 
with  the  polariscope  empty  and 
is  the  point  at  which  the  crystal 
axis  of  the  analyzing  prism  is  at 
right  angles  to  the  plane  of  polar- 
ization, and  the  field  of  vision  is 
uniformly  lighted.  At  either 
side  of  the  zero  point  the  field  is 
unequally  lighted,  as  shown  in 
Fig.  7,  which  indicates  the  ap- 
pearance of  the  field  of  vision  on 
approaching,  on  reaching,  and 
after  passing,  the  zero  point. 
The  upper  figures  of  the  diagram 
represent  the  field  of  a  u  half- 
shade,"  and  the  lower  figures 
those  of  a  "triple-field"  instru- 
ment. If  now  an  active'  sub- 
stance is  placed  as  in  the  tube  T 
between  the  polarizing  and  ana- 
lyzing prisms,  the  latter  must  be  rotated  until  it  stands  at  right 


CARBOHYDRATES  —  GENERAL  METHODS 


81 


FIG.  7.  —  Diagram  of  zero  point  in  reading  polariscope. 


angles  to  the  new  plane,  when  the  appearance  of  the  field  will 
be  the  same  as  that  previously  observed  at  the  zero  point.  The 
position  of  the  scale 
now  shows  directly 
the  number  of  angu- 
lar degrees  through 
which  the  plane  of 
polarization  has  been 
rotated  by  the  active 
substance.  With  this 
polariscope  sodium 
light  is  used. 

For  the  examina- 
tion of  sugar  solu- 
tions in  technical 
analysis  the  "  saccharimeter "  with  the  Ventzke  scale  is  com- 
monly used.  This  instrument,  which  is  made  in  several  forms, 
is  different  from  the  Laurent  polariscope  in  construction  and  in 
that  white  light  is  used ;  and  the  rotation,  instead  of  being 
measured  directly,  is  "  compensated "  by  means  of  quartz 
wedges,  the  thickness  of  quartz  required  to  neutralize  the 

effect  of  the  active  substance  giving 
a  measure  of  the  rotation  produced 
by  the  latter. 

Figure  8  shows  diagrammatically 
the  working  of  the  quartz  compen- 
sator. AB  represents  the  line  of 
vision,  C  and  D  the  wedges  of  dex- 
trorotatory quartz,  and  _Z7  a  section 
of  levorotatory  quartz.  At  the 
FIG.  8.  — Diagram  of  quartz  com-  zero  point  the  combined  thicknesses 

pensator  (after  Rolfe).  rf  Q  an(J  _p  are  equal  tQ  ^  ^^ 

ness  of  E.  When  a  tube  of  (dextrorotatory)  sugar  solution  is 
placed  in  the  instrument  between  the  polarizer  and  the  com- 
pensating device,  the  wedges  are  moved  so  as  to  bring  less 
dextrorotatory  quartz  into  the  line  of  vision  until  the  levorota- 
tory effect  of  E  balances  the  combined  effects  of  (7,  D,  and  the 


82  METHODS  OF  ORGANIC  ANALYSIS 

sugar  solution.  A  scale  attached  to  the  wedge  shows  the 
amount  of  "  compensation "  required  to  balance  the  rotation 
produced  by  the  sugar  and  thus  affords  a  measure  of  the  rota- 
tory power  of  the  sugar.  Instruments  of  this  type  are  usually 
fitted  with  the  Ventzke  scale.  One  degree  on  the  Ventzke 
scale  is  taken  as  equivalent  to  0.3468  degree  of  angular  rota- 
tion. This  method  of  measuring  the  rotation  by  means  of 
quartz  with  white  light  illumination  is  applicable  only  to  such 
active  bodies  as  have  practically  the  same  rotation  dispersion 
as  quartz.  This  is  true  of  aqueous  sugar  solutions,  which  can 
therefore  be  examined  by  either  of  the  instruments  mentioned. 
A  recent  form  of  instrument  of  the  "  saccharimeter  "  type  is 
shown  in  Fig.  9. 


FIG.  9.  —  Recent  form  of  saccharimeter.     (Courtesy  of  Eimer  and  Amend.) 

MEASURE  OF  ROTATING  POWER  —  SPECIFIC  ROTATION 

The  first  organic  substances  to  t)e  studied  by  means  of  polar- 
ized light  were  liquids,  or  solutions  of  known  density.  In 
order  to  reduce  the  observations  to  a  common  basis  for  com- 
parison Biot  introduced  the  following  formula  : 


in  which  [a]  =  the  "  specific  rotating  power  "  ; 


CARBOHYDRATES  —  GENERAL  METHODS         83 

a  =  the  observed  angular  degrees  of  rotation  ; 

I  =  the  length  in  decimeters  of  the  column  of  liquid 

traversed  by  the  polarized  light  ; 
d  =  the  density  of  the  liquid. 

On  extending  the  calculation  to  solids  in  solution  the  above 
formula  becomes 

-,         100  a 


in  which  p  =  the  percentage  of  solid  in  the  solution. 

But  since  d  x  p  equals  the  number  of  grams  of  solid  in  100  cc. 
of  solution,  it  is  simpler  to  write  the  formula 


in  which  c  =  concentration,  i.e.  the  number  of  grams  of  dis- 
solved solid  in  100  cc.  of  the  solution. 

The  value  of  the  specific  rotating  power  [a]  is,  therefore, 
the  number  of  angular  degrees  through  which  a  ray  of  polar- 
ized light  would  be  rotated  in  traversing  one  decimeter  of  a 
solution  of  which  each  cubic  centimeter  contained  one  gram  of 
the  active  substance. 

The  angular  rotation  is  always  directly  proportional  to  the 
length  of  the  column  of  liquid  through  which  the  light  passes. 
It  always  depends  upon  the  wave  length  of  the  light  ray  em- 
ployed. By  measuring  the  rotation  for  different  rays  the 
rotation  dispersion  of  the  substance  is  determined.  Sodium 
light  corresponding  to  the  Fraunhofer  line  D  is  commonly 
used  in  determining  the  rotating  power.  In  many  cases  the 
rotating  power  is  appreciably  influenced  by  temperature.  The 
latter  should  therefore  be  stated  in  giving  the  value  of  [«]. 
Thus  [a]^20  indicates  the  rotating  power  as  measured  with  D 
light  at  20°.  The  concentration  of  the  solution  has  usually  an 
appreciable  influence  upon  the  value  of  [a],  as  is  shown  by  the 
detailed  formulae  for  rotating  power  of  the  more  important 
carbohydrates  given  below. 


84  METHODS  OF  ORGANIC  ANALYSIS 

ROTATORY  POWER  OF  PURE  SUGARS 

To  determine  the  rotatory  power  of  a  pure  sugar  dissolve  10 
grams  in  about  80  cc.  of  water  in  a  100-cc.  flask,  see  that  the 
solution  is  at  room  temperature,  and  note  this  temperature, 
which  should  preferably  be  20°  C.,  fill  to  the  holding  mark,  mix 
the. solution  well,  and  filter  through  a  dry  paper  into  the  polari- 
scope  tube.  The  first  portions  of  the  filtrate  should  be  used 
for  rinsing  the  tube  and  then  rejected.  Finally  fill,  cover,  and 
cap  the  tube,  place  it  in  the  polariscope,  and  observe  the  rotation 
which  it  produces  either  directly  in  angular  degrees  or  by  mul- 
tiplying the  observed  Ventzke  degrees  by  0.3468,  then  calculate 
the  specific  rotatory  power  as  explained  above.  The  details 
and  precautions  regarding  the  handling  of  the  solution  and  use 
of  polariscope  which  are  given  in  the  next  chapter  should  be 
carefully  studied  in  this  connection. 

Following  are  the  detailed  formulae  for  six  of  the  more  impor- 
tant sugars,  showing  corrections  for  concentration  and  tempera- 
ture or  for  concentration  at  some  fixed  temperature : 

Dextrose  (Tollens)  l 

[a]*1*  =  2  52.50  +  0.018796^?  +  0.0005168  j»2. 
Levulose  (Jungfleish  and  Grimbert)  3 

[«y=  -  [101.38 -0.56 £  +  0.1080 -10)]. 
Galactose  (Meissl)  4 

[«]„<  =  83.883  +  0.0785  p  -  0.209 1. 
Sucrose  (Tollens)5 

[a],20  =  66.386  +  0.015035  jt?  -  0.0003986p2. 
Lactose  (Schmoger)6 

[a]^  =  52.53  (constant  for  c  =  2.4  to  40). 
Maltose  (Meissl) 7 

[«]„<  =  140.375  -  0.01837^?  -  0.095 1. 

1  Ber.,  1876,  9,  487,  1531 ;  1884,  17,  2234. 

2  Unless  otherwise  stated  the  rotation  is  to  the  right  (+);  levorotation  is  in- 
dicated by  (-).  3  Compt.  rend,  1888,  107,  390. 

4  J.prakt.  Chem.,  1880,  [2],  22,  97.        5  Ber.,  1877,  10,  1403. 

6  Ber.,  1880,  13,  1922.  7  J.prakt.  chem.,  1882,  [2],  25,  114. 


CARBOHYDRATES  —  GENERAL  METHODS 


85 


At  ordinary  concentrations  and  laboratory  temperatures,  the 
values  for  \_oi]D  are  approximately: 


Dextrose 53 

Levulose     ....  -  [102  -  .56  f] 
Invert  sugar     ...  -  [27.9  -  .32 1] 

Galactose 80.5 

Mannose .     .13 

Arabinose 104 

Xylose 19 


Sucrose 66.5 

Lactose 52.5 

Maltose 138. 

Raffinose 104.5 

Starch  ) 
Glycogen > 
Dextrin  s  160  to  210 


190  to  210 


In  order  to  avoid  confusing  the  determination  of  specific  ro- 
tatory power  with  the  conventional  polariscope  examination  of 
*'  raw  sugar  "  described  in  the  next  chapter,  it  is  well  for  the 
student  at  this  point  to  determine  the  specific  rotatory  power 
of  a  sample  of  sugar  of  known  purity. 

REFERENCES 

I 

ABDERHALDEN  :   Handbuch  der  Biochemisches  Arbeitsmethoden. 

ALLEN  :    Commercial  Organic  Analysis. 

BROWNE  :   Handbook  of  Sugar  Analysis. 

HOPPE-SEYLER  :   Physiologisch  und  Pathologisch-Chemischen  Analyse. 

LANDOLT  :   Optical  Rotation  of  Organic  Substances. 

LIPPMANN  :    Chemie  der  Zuckerarten. 

MAQUENNE  :   Les  Sucres. 

MEYER  and  JACOBSON  :    Organische  Chemie. 

MULLIKEN  :    Identification  of  Pure  Organic  Compounds. 

OPPENHEIMER  :   Handbuch  der  Biochemie. 

ROLFE  :    The  Polariscope  in  the  Chemical  Laboratory. 

TOLLENS  :   Chemie  der  Kohlen hydrate. 

VAUBEL  :   Bestimmung  Organischer  Verbindungen. 

WILEY  :   Agricultural  Analysis,  Vol.  III. 

II 

1905.  LING  and  RENDLE  :   The  Volumetric   Determination  of  Reducing 

Sugars.     Analyst,  30,  182. 

1906.  BROWNE:   The  Analysis  of  Sugar  Mixtures.     J.  Am.  Chem.  Soc.,  28, 

439. 

MUNSON  and  WALKER  :  Unification  of  Reducing  Sugar  Methods. 
/.  Am.  Chem.  Soc.,  28,  663.  See  also  Walker,  same,  29,  541 ;  34, 
202. 


86  METHODS   OF   ORGANIC   ANALYSIS 

1907.  BANG  :    (Determination  of  Reducing  Sugars  by  means  of  Hydroxyl- 

amine).     Biochem.  Z.,  2,  271 ;   Chem.  Abs.,  1,  590. 

BATES  :  A  Quartz  Compensating  Polariscope  with  Adjustable  Sen- 
sibility. U.  S.  Bur.  Standards,  Bui.  4.  Also  Z.  Ver.  Zuckerind., 
58,  105,  821 ;  Chem.  Abs.,  2,  1219,  3165. 

BENEDICT:  The  Detection  and  Estimation  of  Reducing  Sugars. 
J.  Biol.  Chem.,  3,  101. 

BROWNE  and  HALLIGAN  :  Report  on  Sugar.  U.  S.  Dept.  Agr.,  Bur. 
Chem.,  Bui.  105,  p.  116. 

HINKEL  and  SHERMAN  :  Experiments  on  Barfoed's  Acid  Cupric 
Acetate  Solution  as  a  Means  of  Distinguishing  Glucose  from 
Maltose,  Lactose,  and  Sucrose.  J.  Am.  Chem.  Soc.,  29,  1744. 

HUDSON:  Action  of  Acids  and  Bases  on  the  Mutarotation  of  Glu- 
cose. /.  Am.  Chem.  Soc.,  29,  1571. 

MATHEWS  arid  McGuiGAN:  The  Oxidation  of  Sugars  by  Cupric 
Acetic  Acid  Mixtures.  Proc.  Soc.  Exp.  Biol.  Med.,  1907 ;  Chem. 
Abs.,  1,  1780. 

NEF  :  Behavior  of  Sugars  toward  Fehling  Solution  as  well  as  toward 
other  Oxidizing  Agents.  Ann.  Chem.,  357,  214;  Chem.  Abs.,  2, 
799. 

1908.  BUNZEL  :   The   Rate  of   Oxidation  of   Sugars  in  an  Acid  Medium. 

Am.  J.  Physiol.,  21,  23. 

BROWNE  :  Determination  of  Reducing  Sugars  from  Weight  of 
Cuprous  Oxide.  Intern.  Sugar  J.,  10,  537;  Chem.  Abs.,  3,  385. 

KENDALL  and  SHERMAN  :  The  Detection  and  Identification  of  Cer- 
tain Reducing  Sugars  by  Condensation  with  p.  Brombenzylhy- 
drazide.  J.  Am.  Chem.  Soc.,  30,  1451. 

LING  and  JONES  :  (Accuracy  of  Determination  of  Reducing  Sugars) . 
Analyst,  33,  160. 

MEISENHEIMER  :  Behavior  of  Glucose,  Fructose,  and  Galactose 
toward  dilute  Sodium  Hydroxide.  Ber.,  41,  1009. 

1910.  HERSTEIN:    (Historical    Sketch    of    Fehling's    Solution).     J.    Am. 

Chem.  Soc.,  32,  779. 

HUDSON:   Mutarotation.     J.  Am.  Chem.  Soc.,  32,  889. 
NEF  :    Behavior   of  Sugars   toward  Caustic    Alkalies.     Ann.    Chem., 

376,  1. 
SCHLIEPHACKE  :    Mutarotation  of  Maltose.     Ann.  Chem.,  397,  164. 

1911.  BANG:     Preparation    of    Copper    Solutions    for     Sugar     Titration. 

Biochem.  Z.,  32,  443. 
BENEDICT  :   A  Method  for  the  Estimation  of  Reducing  Sugars.     /. 

Biol.  Chem.,  9,  57. 
FISCHER  :    (Determination  of  Rotatory  Power  on  Small  Amounts  of 

Material).     Ber.,  44,  129. 

1912.  KENDALL  :   A  New  Method  for  the  Determination  of  the  Reducing 

Sugars.    J.  Am.  Chem.  Soc.,  34,  317. 


CHAPTER  IV 
Special  Methods  of  Sugar  Analysis 

ANALYSIS  OF  RAW   SUGAR 

POLAKISCOPIC  EXAMINATION 

A  SPECIAL  room  of  even  temperature  which  can  be  darkened 
when  desired,  or  a  dark  screen  which  can  be  placed  around  the 
instrument  on  the  laboratory  table,  should  be  provided  for  the 
polariscopic  examination.  For  illumination  of  the  white-light 
saccharimeter,  a  triple  flame  or  an  Argand  burner  with  incan- 
descent mantle  is  used.  In  taking  readings  the  polariscope 
must  be  brought  only  near  enough  to  the  burner  to  secure  a 
good  illumination  of  the  field,  never  within  less  than  eight 
inches.  As  soon  as  the  reading  is  taken  the  polariscope  should 
be  turned  away  or  the  flame  lowered,  in  order  to  avoid  any 
possible  warming  of  the  instrument. 

Determination  of  the  Zero  Point.  —  The  trough  of  the  sac- 
charimeter being  empty,  set  the  scale  within  a  few  degrees  of 
zero  and  focus  the  eyepiece  so  that  the  field  of  vision  is  clear 
and  the  perpendicular  line  or  band  dividing  it  is  perfectly  dis- 
tinct. Now  rotate  the  milled  head  so  as  to  move  the  zero  point 
of  the  scale  toward  that  of  the  vernier.  When  the  neutral 
point  (the  true  zero  point  for  the  instrument  as  it  stands)  is 
reached,  the  appearance  of  the  entire  field  is  uniform.  Approach 
the  zero  point  first  from  one  side  then  from  the  other,  taking 
the  reading  each  time  as  soon  as  the  entire  field  appears  uni- 
form, until  successive  readings  do  not  differ  by  more  than  0.2° 
on  the  Ventzke  sugar  scale.  The  average  of  six  to  ten  such 
readings  is  taken  as  the  zero  point  in  the  subsequent  work  of 

87 


88  METHODS  OF  ORGANIC  ANALYSIS 

the  day,  unless  the  instrument  should  be  jarred  or  moved,  in 
which  case  the  zero  point  should  be  redetermined. 

Test  with  Pure  Sugar.  —  Weigh  26 l  grams  of  pure  sucrose, 
dissolve  in  water  in  an  accurately  calibrated  100-cc.  flask,  fill 
to  the  mark  at  20°  to  22°,  and  mix  thoroughly  by  shaking,  hold- 
ing the  flask  in  such  a  way  as  not  to  warm  the  solution.  The 
latter  should  have  a  density  very  nearly  1.10  and  should  rotate 
the  plane  of  polarized  light  34. 68°  to  the  right,  giving  a  reading 
of  100.0°  on  the  Ventzke  scale. 

Rinse  the  200-mm.  tube  several  times  with  the  solution,  then 
fill  (by  pouring  through  a  dry  filter,  rejecting  the  first  portions 
of  the  filtrate)  until  the  curved  surface  of  the  liquid  projects 
above  the  open  end  of  the  tube ;  see  that  all  air  bubbles  have 
risen  to  the  surface,  and  then  slide  on  the  cover-glass  horizontally 
in  such  a  manner  that  the  excess  of  liquid  is  carried  over  the 
side,  leaving  the  cover  glass  exactly  closing  the  tube,  with  no 
air  bubbles  beneath  it  and  none  of  the  liquid  upon  its  upper 
surface.  The  cover  glass  being  in  position,  the  tube  is  closed 
by  screwing  on  the  cap.  The  latter  should  be  only  tight 
enough  to  prevent  leakage,  as  any  considerable  pressure  on  the 
glass  plate  may  cause  it  to  become  optically  active.  Look 
through  the  tube  lengthwise  to  be  sure  that  the  cover  glasses 
are  clean  and  dry  and  the  contents  of  the  tube  perfectly  clear. 

Place  the  tube  in  the  trough  of  the  saccharimeter  or  polari- 
scope  and  take  readings  as  in  setting  the  zero  point.  Unless  the 
ends  of  the  polariscope  tube  are  ground  absolutely  parallel, 
the  position  of  the  tube  in  the  instrument  may  influence  slightly 
the  length  of  column  of  sugar  solution  in  the  line  of  vision. 

1  The  value  of  the  Ventzke  scale  was  originally  fixed  by  means  of  pure 
sucrose  solutions  of  1.100  sp.  gr.  at  17.5°  and  it  was  found  that  100  cc.  of  such 
a  solution  contains  26.048  grams  of  sucrose  (weighed  in  air  with  brass  weights). 
For  many  years,  however,  it  has  been  the  custom  of  instrument  makers  to  cali- 
brate the  Ventzke  scale  by  means  of  solutions  containing  26.048  grams  of  sucrose 
in  100  Mohr's  cubic  centimeters.  A  solution  of  very  nearly  the  same  strength 
is  obtained  by  dissolving  26  grams  and  completing  the  volume  at  20°  to  100 
metric  cubic  centimeters  (the  volume  occupied  by  100  grains  of  water  at  4° 
weighed  in  vacuo).  The  latter  proportions  have  been  adopted  by  the  Interna- 
tional Commission  for  Unifying  Methods  of  Sugar  Analysis. 


SPECIAL   METHODS    OF    SUGAR   ANALYSIS  89 

In  order  to  be  sure  that  the  length  whose  rotation  is  measured 
is  the  true  length  of  the  polariscope  tube,  take  two  or  four 
readings,  then  turn  the  tube  two  thirds  over  in  the  trough  of 
the  instrument,  and  after  taking  two  or  four  readings  in  this 
position  turn  the  tube  again  two  thirds  over  in  the  same  direc- 
tion for  the  final  two  or  four  readings.  After  a  little  practice 
it  will  probably  be  found  sufficient  to  take  one  reading  approach- 
ing the  end  point  from  the  right  and  one  from  the  left  in  each 
of  three  positions  of  the  tube,  or  six  readings  in  all.  The  aver- 
age reading  (corrected  for  zero  point)  should  not  differ  from 
100.0°  by  more  than  0.2°  on  the  Yentzke  scale,  nor  from  34°  41' 
on  the  Laurent  polariscope,  by  more  than  5'. 

Polarization  of  the  Raw  Sugar.  —  Mix  the  sample,  weigh  out 
26  grams,  and  dissolve  in  60  to  80  cc.  of  water  in  a  100  cc. 
flask.  When  the  sugar  is  entirely  dissolved,  add  from  1  to 
5  cc.  (according  to  the  nature  of  the  sample ;  only  very  dark 
sugars  should  require  more  than  2  cc.)  of  a  solution  of  basic 
lead  acetate  of  about  1.25  sp.  gr.1  A  decided  excess  of  basic 
acetate  should  be  carefully  avoided.  This  may  be  done  by  add- 
ing the  lead  solution  a  few  drops  at  a  time,  shaking  after  each 
addition,  and  stopping  as  soon  as  another  drop  of  the  solution 
produces  no  further  precipitate. 

After  the  lead  acetate  has  been  added  and  mixed,  add  twice 
its  volume  of  "alumina  cream."2  This  assists  in  the  clarifica- 
tion, precipitates  the  excess  of  lead,  and  facilitates  filtration. 
A  moderate  excess  of  alumina  cream  does  no  harm.  With 
high  grade  sugars  the  use  of  alumina  cream  alone  may  be 
sufficient  for  clarification.  Make  up  to  volume  with  distilled 

1  This  may  be  prepared  by  dissolving  the  solid  basic  salt  or  by  boiling  an  ex- 
cess of  litharge  with  a  strong  solution  of  neutral  lead  acetate. 

2  Prepared  as  follows :    Shake   powdered   commercial   alum  with  water  at 
ordinary  temperature  until  a  saturated  solution  is  obtained.     Set  aside  a  little 
of  the  solution,  and  to  the  residue  add  ammonia,  little  by  little,  stirring  between 
additions,  until  the  mixture  is  alkaline  to  litmus  paper.     Then  drop  in  additions 
of  the  portion  left  aside,  until  the  mixture  is  just  acid  to  litmus  paper.     By  this 
procedure  a  cream  of  aluminium  hydroxide  is  obtained  suspended  in  a  solution 
of  ammonium  sulphate.     This  sulphate  is  advantageous  when  added  after  the 
basic  acetate,  since  it  precipitates  whatever  excess  of  lead  may  be  present. 


90  METHODS  OF  ORGANIC  ANALYSIS 

water,1  shake  well,  and  then  pour  the  whole  solution,  or  as 
much  as  practicable,  on  a  dry  filter.  Reject  the  first  20  or  30 
cc.  of  filtrate  and  then  polarize  the  remainder  as  already  de- 
scribed for  the  pure  sugar  solution.  The  average  reading  of 
the  Ventzke  scale,  corrected  for  the  deviation  of  the  zero  point, 
is  reported  as  "polarization." 

Notes  and  Precautions.  —  Care  must  be  taken  to  avoid  the 
following  errors  due  to  manipulation  :  (1)  change  in  moisture 
content  of  sample  during  weighing,  (2)  change  in  volume  of 
solution  due  to  fluctuations  of  temperature,  (3)  imperfect  mix- 
ing of  solution  after  diluting  to  volume  in  the  graduated  flask, 
(4)  evaporation  during  filtration,  (5)  too  great  compression 
of  cover  glasses  in  closing  the  polariscope  tube. 

Clarification  is  very  important.  All  proteins  are  levorota- 
tory  and  must  be  entirely  removed.  The  solution  must  be 
free  from  turbidity,  but  not  necessarily  free  from  color.  Only 
so  much  of  the  clarifying  agents  should  be  used  as  is  necessary 
to  free  the  solution  from  optically  active  impurities  and  from 
turbidity.  Any  excess  increases  the  error  due  to  the  presence 
of  precipitate  when  the  solution  is  diluted  to  volume.  The 
volume  occupied  by  the  precipitate  varies  between  0.05  and 
1.0  cc.  for  ordinary  raw  sugars.  It  can  be  determined  by 
Scheiber's  method  of  double  dilution,2  in  which  a  duplicate 
determination  is  made  in  a  flask  of  twice  the  volume,  or  by 
determining  the  weight  and  density  of  the  precipitate  as  rec- 
ommended by  Sachs.3  The  latter  method  is  preferred  by 
Wiechmann  4  and  Home,  5  each  of  whom  determined  the  volume 
of  the  precipitate  for  a  number  of  raw  sugars  from  different 
localities.  Home  has  found  (loc.  cit.)  that  the  error  can  be 
almost  entirely  avoided  by  clarifying  with  anhydrous  basic 
acetate  after  the  sugar  solution  has  been  diluted  to  volume, 
and  this  method  of  clarification,  after  extended  investigation 

1  If  frothing  interferes  at  this  point,  add  two  or  three  drops  of  ether. 

2  Z.  Vereins.  Biibenzucker  Industrie,  1875,  25,  1054. 

3  /bid.,  1880,  50,  229.     See  also  the  paper  by  Home. 

4  Ibid.,  1903,  [n.  f.]  40,  498 ;  Abs.  J.  Chem.  Soc.,  1903,  84,  ii,  699. 
6  J.  Am.  Chem.  Soc.,  1904,  26,  186. 


SPECIAL   METHODS   OF   SUGAR   ANALYSIS  91 

and  discussion,  has  now  been  adopted  by  the  International 
Commission  on  a  parity  with  the  usual  method.  According 
to  Browne,  the  amount  of  dry  acetate  used  should  not  exceed 
0.5  gram. 

Regulations  of  the  International  Commission 

The  following  resolutions, 1  adopted  by  the  International 
Commission  for  the  Unification  of  Sugar  Analysis,  in  1900, 
have  since  been  generally  accepted. 

1.  In  general,  all  sugar  tests  shall  be  made  at  20°  C.2 

2.  The  graduation  of  the  saccharimeter  shall  be  made  at  20° 
C.     Twenty-six  grams  of  pure  sugar,  dissolved  in  water,  and 
the  volume  made  up  to  100  metric  cubic  centimeters,  or  during 
the  period  of   transition  26.048  grams   of   pure  sugar  in  100 
Mohr  cubic  centimeters,  all  weighings  to  be  made  in  air  with 
brass  weights,  the  completion  of  the  volume  and  the  polariza- 
tions to  be   made  at  20°  on  an  instrument  graduated  at  20°, 
should  give  an  indication  of  100  on  the  scale  of  the  saccharim- 
eter.      For    countries    where  temperatures  are  usually  higher 
than  20°,  it  is  permissible  that  saccharimeters  be  graduated  at 
30°,  or  any  other  suitable  temperature,  under  the  conditions 
specified   above,  providing  that   the  analysis  of  the  sugar  be 
made  at  the  same  temperature  —  that  is,  that  the  volume  be 
completed    and    the    polarization    made    at    the    temperature 
specified. 

3.  Preparation  of  pure  sugar:    Purest  commercial  sugar  is 
to  be  further  purified  in  the  following  manner:    A  hot  satu- 
rated aqueous  solution  is  prepared  and  the  sugar  precipitated 
with  absolute  ethyl  alcohol ;  the  sugar  is  carefully  spun  in  a 

1  U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bull.  73,  p.  58.     J.  Am.  Chem.  Soc., 
1901,  23,  59. 

2  In  polarizing  pure  sucrose  with  quartz  wedge  saccharimeter  there  is  a  falling 
off  in  polarization  of  about  0.03°  Ventzke  for  each  degree  C.  that  the  tempera- 
ture rises  above  20°.     Crude  sugars,  however,  may  contain  enough  levulose  so 
that  their  polariscope  readings  are  increased  by  a  rise  of  temperature.     Browne 
has  clearly  shown  that  temperature  errors  can»not  be  compensated  by  corrections 
based  on  pure  sucrose,  but  must  be  avoided  by  actually  polarizing  at  or  near 
20°  C. 


92  METHODS    OF   ORGANIC   ANALYSIS 

small  centrifugal  machine  and  washed  in  the  latter  with  abso- 
lute alcohol.  The  sugar  thus  obtained  is  redissolved  in  water, 
the  saturated  solution  again  precipitated  with  alcohol  and 
washed  as  above.  The  product  of  the  second  crop  of  crystals 
is  dried  between  blotting  paper  and  preserved  in  glass  vessels 
for  use.  The  moisture  still  contained  in  the  sugar  is  deter- 
mined and  taken  into  account  when  weighing  the  sugar  which 
is  to  be  used. 

The  committee  further  decided  that  central  stations  shall 
be  designated  in  each  country  which  are  to  be  charged  with 
the  preparation  and  distribution  of  chemically  pure  sugar. 
Wherever  this  arrangement  is  not  feasible,  quartz  plates,  the 
values  of  which  have  been  determined  by  means  of  chemically 
pure  sugar,  shall  serve  for  the  control  of  the  saccharimeters. 

The  committee  further  decided  that  the  above  control  of 
quartz  plates  by  means  of  chemically  pure  sugar  should,  as  a 
rule,  apply  only  to  the  central  stations  which  are  to  test  the 
correctness  of  saccharimeters  ;  for  those  who  execute  commer- 
cial analyses,  the  repeated  control  of  the  instruments  is  to  be 
accomplished,  now  as  before,  by  quartz  plates. 

4.  In   effecting  the   polarization   of   substances   containing 
sugar,  half-shaded  instruments,  or  triple  field,  only  are  to  be 
employed. 

5.  During  the  observation  the  apparatus  must  be  in  a  fixed 
position  and  so  far  removed  from  the  source  of  light  that  the 
polarizing  nicol  is  not  warmed. 

6.  Sources  of  light  may  be  gas,  triple  burner  with  metallic 
cylinder,  lens,  and  reflector ;  gas  lamp  with  Auer  (Welsbach) 
burner  ;  electric  lamp  ;  petroleum  duplex  lamp ;  sodium  light. 
Several  readings  are  to  be  made  and  the  mean  thereof  taken, 
but  any  one  reading  must  not  be  neglected. 

7.  In  making  a  polarization  the  whole  normal  weight  for  100 
cubic  centimeters  is  to  be  used,  or  a  multiple  thereof  for  any 
corresponding  volume. 

8.  As   clarifying  and  decolorizing   reagents  there  may  be 
used :   (a)  subacetate  of  lead,  (3  parts  by  weight  of  acetate  of 
lead,  one  part  by  weight  of  oxide  of  lead,  10  parts  by  weight 


SPECIAL   METHODS   OF   SUGAR   ANALYSIS  93 

of  water)  ;  (5)  alumina  cream ;  (<?)  concentrated  solution  of 
alum.  Boneblack  and  decolorizing  agents  are  to  be  excluded. 
9.  After  bringing  the  solution  exactly  to  the  mark,  at  the 
proper  temperature,  and  after  wiping  out  the  neck  of  the  flask 
with  filter  paper,  all  of  the  well-shaken  clarified  sugar  solution 
is  poured  upon  a  dry  rapidly  acting  filter.  The  first  portions 
of  the  filtrate  are  to  be  rejected  and  the  rest,  which  must  be 
perfectly  clear,  used  for  polarization. 

Use  of  Dichromate  Solution  as  Light  Filter 

As  explained  in  the  last  chapter  the  use  of  white  light  in 
polarizing  sugar  solutions  with  the  quartz  compensator  is  based 
upon  the  observation  that  the  rotation  dispersion  is  the  same 
for  sugar  solutions  as  for  quartz.  While,  this  is  practically 
true,  it  is  not  absolutely  correct  at  high  rotations,  even  with 
sucrose  solutions,  since  the  dispersive  powers  of  sucrose  and 
quartz  are  slightly  different  at  the  blue  end  of  the  spectrum. 
This  difference  results  in  a  slight  disparity  of  tint  which  may 
give  rise  to  apparent  differences  of  rotation  when  the  same  sugar 
solution  is  read  by  different  observers  or  even  by  the  same  ob- 
server with  white  light  from  different  sources.  Browne, 1  con- 
firming and  extending  the  earlier  work  of  Schonrock,  finds  that 
this  source  of  error  is  by  no  means  negligible  even  when  work- 
ing with  ordinary  sugar  solutions,  and  should  always  be  guarded 
against  by  passing  the  light  through  a  solution  of  dichromate  to 
absorb  the  blue  and  violet  rays  which  cause  the  greatest  amount 
of  rotation  dispersion.  Saccharimeters  of  recent  manufacture 
have  a  short  tube  or  cell  through  which  the  light  passes  after 
entering  the  instrument  and  before  reaching  the  solution.  This 
tube  if  3  cm.  long  should  be  filled  with  a  three  per  cent  solution 
of  potassium  dichromate  ;  if  1.5  cm.,  with  a  6  per  cent  solution. 
The  German  Reichsanstalt  and  the  U.  S.  Bureau  of  Standards 
now  use  such  dichromate  cells  in  testing  quartz- wedge  saccha- 
rimeters.  Before  using  a  new  instrument  or  one  which  has  been 
out  of  use  for  a  time,  the  dichromate  cell  should  be  examined 

!U.  S.  Dept.  Agr.,  Bur.  Chem.,  Bui.  122,  p.  221. 


94  METHODS   OF   ORGANIC    ANALYSIS 

to  see  that  it  is  properly  filled.     In  class-work  this  will  be 
done  by  the  instructor  rather  than  the  individual  student. 

In  working  with  substances  such  as  commercial  glucose, 
malt  products,  and  dextrins  (whose  rotation  dispersion  is 
greater  than  that  of  cane  sugar),  Browne  finds  it  desirable  to 
double  the  usual  strength  of  the  dichromate  solution. 

Relation  of  Polarization  to  Percentage  of  Sucrose 

In  the  absence  of  other  active  substances,  the  reading  of  the 
Ventzke  scale  as  obtained  above  shows  the  percentage  of  sucrose 
in  the  sample.  In  commercial  raw  sugar  the  only  other  sugars 
likely  to  be  present  are  dextrose  and  levulose.  If  the  total 
amount  of  these  reducing  sugars,  as  shown  by  Fehling's  solution, 
is  small,  the  value  of  the  sample  is  sufficiently  indicated  by  the 
polarization,  the  invert  sugar,  arid  the  amounts  of  moisture  and 
ash. 

Since,  however,  the  "  reducing  sugar  "  contains  dextrose  and 
levulose  in  unknown  proportions,  the  determination  of  "reduc- 
ing power  "  does  not  give  the  necessary  data  for  estimating  the 
percentage  of  sucrose  in  the  sample.  The  percentage  of  sucrose 
can,  however,  be  determined  in  the  presence  of  dextrose  and 
levulose  by  observing  the  change  in  rotation  produced  by  hy- 
drolysis. This  is  unaffected  by  the  presence  of  monosaccha- 
rides,  since  the  latter  cannot  be  hydrolyzed. 

The  following  method  is  based  on  this  principle. 

Clerget's  Method  for  Sucrose 

A  pure  sucrose  solution  polarizing  100°  on  the  Ventzke  scale 
at  a  temperature  of  20°,  will,  after  hydrolysis,  polarize  about 

—  33°  on  the  same  scale  at  the  same  temperature.     It  is  this 
change  of  rotation  from  right  to  left  which  gives  rise  to  the 
terms  "inversion"  for  hydrolysis,  and  "invert  sugar"  for  the 
mixture  of   equal   parts   of    dextrose   and   levulose   produced. 
Since  the  levorotatory  power  of  levulose,  and  hence  of  invert 
sugar,  decreases  rapidly  with  increase  of  temperature,  the  reading 

—  33°  would  be  found  only  at  20°.     An  increase  of  1°  in  temper- 


SPECIAL   METHODS    OF   SUGAR   ANALYSIS  95 

ature  causes  a  decrease  of  practically  0.5  degree  Ventzke  in  the 
levorotation  of  the  invert  sugar,  and  at  about  87°  the  reading 
becomes  zero.  The  change  in  rotation  produced  by  hydrolysis 
in  pure  sucrose  solution  is,  therefore,  about  143  —  0.5  £,  in  which 
t  is  the  temperature  at  which  the  readings  are  taken. 

To  estimate  the  sucrose  in  an  unknown  sample,  dissolve  26 
grams,  clarify  if  necessary,  dilute  to  100  cc.,  filter,  and  polarize 
as  usual  at  20°.  The  remainder  of  the  solution  is  freed  from 
lead  if  necessary  by  treating  with  anhydrous  sodium  carbonate 
or  sulphate,  or  with  potassium  oxalate,  followed  by  filtration 
through  dry  paper.  To  50  cc.  of  the  solution  add  25  cc.  of 
water  and  then  add,  in  small  portions  with  constant  shaking, 
5  cc.  of  concentrated  hydrochloric  acid  (1.196  specific  gravity). 
Place  the  flask  in  a  bath  of  water  kept  at  70°.  The  tempera- 
ture of  the  solution  in  the  flask  should  reach  67°  to  69°  in  2J  to 
3  minutes  and  is  then  maintained  at  a  temperature  as  nearly 
69°  as  possible  for  7  to  7|  minutes,  making  a  total  time  of 
heating  of  10  minutes;  or  the  solution  after  adding  the  acid 
may  be  set  aside  at  a  temperature  of  20°  to  24°  C.  for  24  hours. 
The  sucrose  having  been  thus  hydrolyzed,  cool  the  solution,  if 
necessary,  to  20°  C.  (or  to  room  temperature),  dilute  to  100  cc., 
mix  the  solution  well  and  polarize,  preferably  in  a  water- 
jacketed  200-mm.  tube  with  a  side  tube  through  which  the  exact 
temperature  of  the  solution  at  the  time  of  polarization  may 
be  determined.  Since  50  cc.  of  the  sugar  solution  was  diluted 
for  hydrolysis  and  finally  made  up  to  100  cc.,  this  second  reading 
must  be  doubled  to  obtain  the  so-called  "invert  polarization." 
The  difference  between  the  first  polarization  and  the  "invert" 
polarization,  multiplied  by  100  and  divided  by  the  difference 
which  pure  sucrose  would  show  under  the  same  conditions, 
gives  the  percentage  of  sucrose;  or 

100  (P-I) 
142.66-  0.5 1* 

in  which  s  =  percentage  of  sucrose,  P  the  original  polarization, 
I  the  "invert"  polarization,  and  t  the  temperature  in  degrees 
centigrade. 


96  METHODS  OF  ORGANIC  ANALYSIS 

The  method  is  applicable  to  the  determination  of  sucrose  in 
the  presence  of  any  other  sugar,  or  mixture  of  sugars,  which  is 
not  measurably  affected  by  the  prescribed  treatment  with  acid. 

DETERMINATION  OF  REDUCING  SUGAR 

Usually  the  reducing  power  of  the  raw  sugar  is  determined 
volume trically,  as  described  in  the  preceding  chapter,  the 
results  being  calculated  as  percentage  of  invert  sugar.  For  the 
determination  dissolve  5  grams  of  the  sample  if  dark,  or  10 
grams  if  light,  dilute  to  100  cc.,  and  filter  through  dry  paper. 
If  the  preliminary  test  shows  the  solution  to  be  too  strong  or 
too  weak,  prepare  a  fresh  solution  for  the  final  determination. 
When  the  volumetric  method  is  carefully  carried  out,  the  results 
are  sufficiently  exact  for  all  ordinary  work.1  For  the  deter- 
mination of  very  small  amounts  of  invert  sugar  the  method 
introduced  by  Herzfeld,2  and  recommended  by  the  International 
Commission,3  is  probably  the  most  accurate. 

Herzf eld's  Gravimetric  Method 

Dissolve  20  grams  of  sample  in  80  cc.  of  water,  clarify  with 
basic  lead  acetate,  and  precipitate  the  excess  of  lead  by  sodium 
carbonate  (avoiding  more  than  a  slight  excess)  or  by  neutral 
potassium  oxalate  ;  4  dilute  to  100  cc.,  mix  thoroughly,  and 
filter  through  dry  paper.  The  filtrate  must  be  perfectly  clear. 
In  a  beaker  of  250  cc.  capacity,  place  50  cc.  of  the  undiluted 
"mixed  Fehling  reagent"  (see  Fehling's  volumetric  method) 
and  50  cc.  of  the  clarified  sugar  solution ;  heat  at  such  a  rate 
that  the  mixture  boils  in  about  four  minutes,  and  boil  for  ex- 
actly two  minutes.  Add  100  cc.  of  cold,  recently  boiled,  dis- 
tilled water,  filter  immediately,  and  find  the  amount  of  copper 

1  If  a  gravimetric  method  is  preferred,  use  that  of  Meissl  and   Hiller,  as 
adopted  by  the  Official  Agricultural  Chemists :    Bui.  107,   Bur.    Chem.,  U.  S 
Dept.  Agriculture. 

2  Z.  Vereins  Rubenzucker  Industrie,  1886,  6. 

3  J.  Am.  Chem.  Soc.,  1901,  23,  64. 

*  Sawyer :  J.  Am.  Chem.  Soc.,  1904,  26,  1631. 


SPECIAL   METHODS   OF   SUGAR   ANALYSIS 


97 


reduced  in  one  of  the  ways  described  under  Defren's  method 
in  the  preceding  chapter.  The  following  table  shows  the 
corresponding  percentage  of  invert  sugar. 

TABLE  8.  —  HERZFELD'S  TABLE  FOR  THE  DETERMINATION  OF  INVERT 
SUGAR  IN  MATERIALS  CONTAINING  1  PER  CENT  OR  LESS  OF  INVERT 
SUGAR  AND  A  HIGH  PERCENTAGE  OF  SUCROSE 


Copper  reduced 
by  10  grains 
of  material 
Milligrams 

Iiivert  sugar 
Per  cent 

Copper  reduced 
by  10  grams 
of  material 
Milligrams 

Invert  sugar 
Per  cent 

Copper  reduced 
by  10  grams 
of  material 
Milligrams 

Invert  sugar 
Per  cent 

50 

0.05 

120 

0.40 

190 

0.79 

55 

0.07 

125 

0.43 

195 

0.82 

60 

0.09 

130 

0.45 

200 

0.85 

65 

0.11 

135 

0.48 

205 

0.88 

70 

0.14 

140 

0.51 

210 

0.90 

75 

0.16 

145 

0.53 

215 

0.93 

80 

0.19 

150 

0.56 

220 

0.96 

85 

0.21 

155 

0.59 

225 

0.99 

90 

0.24 

160 

0.62 

230 

1.02 

95 

0.27 

165 

0.65 

235 

1.05 

100 

0.30 

170 

0.68 

240 

1.07 

105 

0.32 

175 

0.71 

245 

1.10 

110 

0.35 

180 

0.74 

115 

0.38 

185 

0.76 

DETERMINATION  OF  MOISTURE  AND  ASH 

Dry  about  2  grams  of  sample  in  a  flat-bottomed  platinum 
dish  in  the  boiling  water  oven  until  the  loss  in  weight  on  heat- 
ing for  one  hour  does  not  exceed  0.10  per  cent.  A  perfectly 
constant  weight  cannot  be  expected  if  invert  sugar  is  present, 
since  levulose  is  slowly  decomposed  by  heating  at  100°  in  the 
air.  Calculate  the  loss  of  weight  as  moisture. 

Moisten  the  residue  with  a  few  drops  of  concentrated  sul- 
phuric acid  and  burn  to  whiteness.  Weigh,  and  report  the 
result  as  "sulphated  ash."  The  true  ash  is  considered  by 
some  as  nine  tenths  and  by  others  as  four  fifths  of  the  "sul- 
phated ash." 


98  METHODS  OF  ORGANIC  ANALYSIS 

OFFICIAL  METHODS  OF  TESTING  SUGARS 

Methods  of  the  Association  of  Official  Agricultural  Chem- 
ists :  Bui.  107,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 

Regulations  governing  the  sampling  and  classification  of 
imported  sugars  and  molasses :  Treasury  Department  Docu- 
ment No.  2470. 

German  official  methods  :  Z.  Nahr.  Grenussm.,  1903,  6,  1059  ; 
Z.  anal.  Chem.,  1903,  42  (Appendix  to  Nos.  9  and  10). 

DETERMINATION   OF   SUCROSE  IN   BEETS   AND   CANE 

Sucrose  is  the  only  sugar  found  in  appreciable  quantity  in 
the  fresh  juice  of  healthy  sugar  beets.  Sugar  cane  juice  con- 
tains a  small  amount  of  reducing  sugar  which  is  usually  con- 
sidered to  be  invert  sugar,  but  which,  as  Browne  has  shown, 
may  contain  dextrose  and  levulose  in  varying  proportions. 
In  the  routine  testing  of  beet  and  cane  juices,  the  polarization 
of  a  properly  clarified  sample  is  considered  as  showing  the 
amount  of  sucrose  present. 

Beets  after  being  thoroughly  cleaned  (the  loss  of  weight  in 
cleaning  being  determined  and  reported  as  "  tare  ")  are  reduced 
to  a  fine  pulp  by  rasping  or  grinding.  This  pulp  is  then  treated 
in  one  of  three  ways:  (1)  separating  the  insoluble  matter  by  pres- 
sure and  polarizing  the  juice,  (2)  extracting  with  alcohol,  (3) 
extracting  with  water.  The  first  method  introduces  errors  from 
imperfect  separation  of  the  juice,  the  second  is  longer  and  more 
expensive  than  the  third,  which  is  now  generally  used.  If,  how- 
ever, the  pulp  be  treated  with  water  and  filled  to  volume  in  a 
graduated  flask,  as  in  the  analysis  of  raw  sugar,  it  is  very  difficult 
to  expel  the  air  bubbles  entirely  from  the  mixture  of  pulp  and 
water.  The  following  method,  described  by  Sachs,1  avoids  this 
inconvenience  and  has  been  found  satisfactory  for  rapid  work: 
Weigh  26  grams  of  the  pulp  in  a  beaker  and  add  177  cc.  of  water 
containing  5  cc.  of  the  usual  solution  of  basic  lead  acetate ;  shake 
or  stir  for  three  minutes  ;  filter,  and  polarize  the  filtrate.  Twice 

1  Paper  read  before  the  Second  International  Congress  of  Applied  Chemistry, 
Paris,  1896  ;  quoted  by  Hiltner  and  Thatcher,  J.  Am.  Chem.  Soc.,  1901,  23,  300. 


SPECIAL   METHODS   OF   SUGAR   ANALYSIS  99 

the  reading  of  the  Ventzke  scale  is  approximately  the  percentage 
of  sucrose  in  the  sample.  This  method  assumes  that  26  grams 
of  beet  pulp  contain  23  cc.  of  juice,  making  the  total  volume  of 
liquid  in  the  mixture  200  cc.  Many  German  sugar  chemists  hold 
that  extraction  with  water  gives  high  results  because  the  beet 
contains  substances  other  than  sucrose  which  are  soluble  in 
water  and  rotate  polarized  light  to  the  right.  According  to 
Sachs,  however,  these  substances  are  pectin-like  bodies  and  are 
completely  precipitated  by  basic  lead  acetate.  Trowbridge l  also 
found  that  the  results  obtained  by  digestion  in  water  were 
practically  identical  with  those  of  the  alcohol  extraction 
method. 

In  sampling  sugar  cane  for  analysis,  the  canes  are  usually 
cut  into  thin  oblique  chips,  "  cosettes,"  by  means  of  rotating 
knives.  These  cosettes  may  be  further  reduced  by  passing 
through  a  sausage  cutter  or  a  special  shredding  machine.  It 
is  not  feasible,  however,  to  reduce  the  cane  to  as  fine  a  pulp 
as  is  obtained  from  sugar  beets.  For  this  reason  more  time 
and  a  higher  temperature  must  be  used  to  insure  complete 
extraction  of  the  sucrose.  According  to  Wiley,2  26  grams  of 
the  chips  or  pulp  are  weighed  into  a  flask  graduated  at  102.6 
cc.,  this  graduation  being  based  on  the  assumption  that  the 
cane  contains  ten  per  cent  of  insoluble  matter  of  a  specific 
gravity  about  equal  to  that  of  the  solution.  The  flask  is 
nearly  filled  with  water,  and  warmed  on  a  water  bath  for  one 
hour  with  frequent  shaking;  then  filled  a  little  above  the  mark 
and  warmed  for  ten  minutes  longer  with  frequent  shaking. 
The  flask  is  now  cooled,  the  volume  adjusted  if  necessary,  and 
the  liquid  filtered  through  dry  paper.  Fifty  cubic  centimeters 
of  filtrate  are  collected  in  a  55-cc.  flask,  clarified  with  basic 
acetate,  filled  to  the  mark,  shaken,  filtered,  and  the  filtrate 
polarized  in  a  220-mm.  tube. 

For  further  information  on  the  analysis  of  sugar  beets 
and  sugar  cane,  the  following  books  and  papers  may  be  con- 
sulted : 

1  J.  Am.  Chem.  floe.,  1901,  23,  300. 

2  Agricultural  Analysis,  Vol.  Ill,  p.  238. 


100  METHODS    OF    ORGANIC    ANALYSIS 

Wiley:   Agricultural  Analysis,  Vol.  Ill,  Part  III. 

Spencer:  Handbook  for  Beet  Sugar  Chemists.  Handbook  for 
Sugar  Manufacturers. 

Lippmann  and  Pulvermacher:  Lunge's  Chemisch-technische 
Untersuchungsmethoden. 

Friihling:  Anleitung  zur  Untersuchung  fiir  die  Zucker- 
industrie. 

Trowbridge:  Notes  on  Sugar  Beets,  J.  Am.  Chem.  iSoc.,  1901, 
23,  216. 

Hiltner  and  Thatcher:  An  improved  Method  for  the  Rapid 
Estimation  of  Sugar  in  Beets,  J.  Am.  Chem.  Soc.,  1901,  23,  299, 
863. 

DENSITY  AND  PURITY   OF   SUGAR  SOLUTIONS 

The  relations  between  density  and  percentage  in  pure  sugar 
solutions  have  been  very  carefully  determined,  and  hydrometers 
are  specially  made  for  sugar  testing  which  are  graduated  to  give 
at  a  standard  temperature  (usually  17.5°  C.)  a  direct  reading 
of  the  per  cent  of  sugar  present  in  the  solution.  These  are 
usually  known  as  Brix  (sometimes  as  Balling)  hydrometers 
or  "spindles."  Brix  hydrometers  often  have  thermometers 
attached,  and  sometimes  carry  scales  for  reading  temperature 
corrections  directly  from  the  instrument.  Detailed  tables  for 
temperature  corrections  of  Brix  hydrometers  and  for  conversion 
of  specific  gravity  in  Brix  reading  (percentage  of  sucrose)  or 
the  reverse  are  given  in  technical  works  on  sugar  analysis  such 
as  those  of  Rolfe  and  Spencer. 

Beet  and  cane  juices,  although  containing  other  solids  than 
sucrose,  are  often  tested  with  the  Brix  hydrometer  and  the 
reading  taken  as  an  approximate  measure  of  the  total  solids 
present. 

The  polarization  multiplied  by  100  and  divided  by  the  Brix 
reading  is  sometimes  called  the  "  quotient  of  purity  "  of  the  juice. 
This  of  course  is  not  exactly  the  percentage  of  sucrose  in  the 
solids,  because  in  impure  solutions  the  polarization  may  not  be  an 
exact  measure  of  the  sucrose,  and  the  Brix  hydrometer  reading 
is  not  an  exact  measure  of  the  percentage  of  total  solids  ;  it  is, 


SPECIAL    METHODS    OF    SUGAR   ANALYSIS'  101 

however,   an  indication  of  purity  which  is  of  much  value  in 
controlling  the  operations  of  the  sugar  house. 

In  recent  years  the  immersion  refractometer  has  been  con- 
siderably used  in  sugar  house  control.  Tables  have  been  con- 
structed showing  the  percentages  of  sugar  corresponding  to  each 
degree  on  the  refractometer  scale,  and,  with  the  aid  of  these, 
refractometer  readings  of  sugar  solutions  or  of  cane  or  beet  juices 
are  readily  translated  into  terms  comparable  with  the  Brix 
hydrometer  readings,  though  of  course  any  given  impurity  may 
not  indicate  exactly  the  same  percentage  of  sugar  by  refraction 
that  it  would  by  specific  gravity. 

IDENTIFICATION   AND   ANALYSIS   OF   "UNKNOWN"  SUGARS 

Given  an  "  unknown  "  sugar  in  an  approximately  pure  state 
or  an  unknown  mixture  of  two  or  three  such  sugars,  one  should 
find  little  difficulty  in  making  out  an  indentification  and  analy- 
sis from  the  methods  and  data  which  have  been  given  in  this 
and  the  preceding  chapter. 

It  is  well  to  begin  by  determining  the  specific  rotatory  power 
and  the  reducing  power  (calculating  the  latter  in  percentage  of 
the  reducing  power  of  pure  dextrose  as  explained  in  the  last 
chapter)  and  by  performing  an  osazone  test  under  the  standard 
conditions,  noting  the  time  of  appearance  and  the  abundance  of 
the  osazone  precipitate,  if  any  appears  in  the  hot  solution,  and 
whether  any  further  precipitation  of  osazone  occurs  as  the  so- 
lution cools.  Keeping  in  mind  the  influence  of  quantity  of  re- 
acting sugar  and  of  other  sugars  present  on  the  formation  and 
precipitation  of  the  osazone  and  comparing  the  result  obtained 
in  the  osazone  test  with  the  rotatory  and  reducing  powers,  one 
can  usually  judge  from  these  data  the  sugars  likely  to  be  present 
in  the  sample.1 

1This  judgment  may  be  aided  by  certain  superficial  observations,  e.g.  if  the 
10  grams  taken  for  the  determination  of  rotatory  power  are  difficultly  soluble  in 
60  cc.  of  water  the  presence  of  considerable  lactose  may  be  suspected  ;  while  if 
the  solution  readily  undergoes  fermentation  with  yeast,  it  is  probable  that  glucose, 
levulose,  or  maltose  is  present,  though  it  is  to  be  remembered  that  ordinary  yeast 
also  acts  on  sucrose,  first  hydrolyzing  it  to  dextrose  and  levulose  and  then  fer- 


102  METHODS    OF   ORGANIC   ANALYSIS 

If  only  two  sugars  are  present  in  the  mixture,  the  two  quan- 
titative determinations  already  made  will  suffice  for  the  calcu- 
lation of  the  percentage  of  each.  For  example,  if  the  mixture 
consists  of  dextrose  and  lactose,  then 

100  a  =  53  d  +  52.5  I, 


in  which  a  is  the  specific  rotatory  power  of  the  mixture,  TTthe 
cupric  reducing  power  of  the  mixture  (calculated  in  percent- 
age of  the  reducing  power  of  an  equal  weight  of  pure  dextrose 
as  explained  before),  d  the  percentage  of  dextrose,  and  I  the 
percentage  of  lactose.  Such  a  pair  of  simultaneous  equations, 
one  expressing  rotatory  and  the  other  reducing  power,  will 
serve  for  any  two  of  the  common  sugars  at  least  one  of  which 
reduces  Fehling's  solution. 

If  three  sugars  are  present  in  the  mixture,  it  is  evident  that 
a  third  quantitative  determination  (to  give  the  data  for  a  third 
equation)  will  be  required.  If  sucrose  is  one  of  the  three,  it 
may  be  determined  by  the  Clerget  method,  even  in  the  pres- 
ence of  maltose  or  lactose,  with  at  least  approximately  correct 
results,  since  the  acid  treatment  prescribed  for  the  hydrolysis 
of  the  sucrose  does  not  hydrolyze  maltose  nor  lactose  to  any 
great  extent.  The  analysis  of  a  mixture  of  three  sugars  other 
than  sucrose  is  less  easy  and  will  usually  be  less  accurate. 
Levulose,  if  present,  may  be  determined  by  the  decrease  of 
its  levorotatory  power  on  polarizing  at  a  known  high  tempera- 
ture ;  lactose,  galactose,  or  raffinose  may  be  estimated  from  the 
yield  of  mucic  acid  ;  maltose  may  be  quantitatively  hydrolyzed 
as  described  in  the  next  chapter.  In  some  cases  it  may  be 
feasible  to  determine  the  moisture,  the  ash,  and  any  organic 
constituents  other  than  the  sugars,  subtract  the  sum  of  the 
percentages  of  all  these  impurities  from  100,  and  take  the 
remainder  as  the  sum  of  the  percentages  of  the  different 

menting  these.  [In  laboratories  where  pure  cultures  of  different  yeasts  are 
available  the  difference  of  behavior  of  different  yeasts  toward  the  sugars  can  be 
made  use  of  for  analytical  purposes,  but  in  most  chemical  laboratories  such  pure 
cultures  are  not  at  hand.  ] 


SPECIAL   METHODS   OF   SUGAR  ANALYSIS  103 

sugars  present ;  but,  as  a  rule,  this  should  be  resorted  to  only 
for  purposes  of  verification,  and  not  as  a  means  of  obtaining 
one  of  the  original  simultaneous  equations. 

REFERENCES 

I 

ALLEN  :  Commercial  Organic  Analysis,  Vol.  I. 

BROWNE  :  Handbook  of  Sugar  Analysis. 

FRUHLING  :  Anleitung  ziir  Untersuchung  fiir  die  Zuckerindustrie. 

LANDOLT  (transl.  by  Long)  :  Optical  Rotation  of  Organic  Substances. 

LEACH  :  Food  Inspection  and  Analysis. 

LIPPMANN  :  Chemie  der  Zuckerarten. 

LUNGE  :  Chemisch-technische  Untersuchungsmethoden. 

ROLFE  :  The  Polariscope  in  the  Chemical  Laboratory. 

SPENCER  :  Handbook  for  Beet  Sugar  Chemists. 

Handbook  for  Sugar  Manufacturers  (Cane  Sugar). 
WILEY  :  Agricultural  Analysis. 

Periodicals  devoted  to  Sugar 

American  Sugar  Industry  and  Beet  Sugar  Gazette. 

Bulletin  de  1'Association  des  Chimistes  de  Sucrerie  et  de  Distillation. 

Centralblatt  fiir  die  Zuckerindustrie. 

Die  Deutsche  Zuckerindustrie. 

International  Sugar  Journal. 

Louisiana  Sugar  Planter  and  Sugar  Manufacturer. 

Oesterreichisch-Ungarische  Zeitschrift  fiir  Zuckerindustrie. 

Neue  Zeitschrift  fiir  Riibenzuckerindustrie. 

Zeitschrift  des  Vereins  der  deutschen  Zuckerindustrie. 

Zeitschrift  fiir  Zuckerindustrie  in  Bohmen. 

II 

1895.  WEBER  and  MCPHERSON  :  On  the  Determination  of  Cane  Sugar  in 

the  Presence  of  Commercial  Glucose.    /.  Am.  Chem.  Soc.,  17,  312. 

1896.  WILEY  :  On  the  Estimation  of  Levulose  in  Honeys  and  Other  Sub- 

stances.    J.  Am.  Chem.  Soc.,  18,  81. 

1902.  BOYDEN:  On  the  Quantitative   Separation  of  Maltose  and  Lactose. 

/.  Am.  Chem.  Soc.,  24,  993. 

1903.  LEACH  :  The    Determination   of    Commercial   Glucose  in  Molasses, 

Syrups,  and  Honey.     J.  Am.  Chem,  Soc.,  25,  982. 

1904.  HARRISON  :  Determination  of  Sucrose  and  Lactose  in  Condensed  Milk. 

Analyst,  29,  248. 


104  METHODS   OF   ORGANIC   ANALYSIS 

HORTVET:  The  Chemical  Composition  of  Maple  Syrup  and  Maple 
Sugar,  Methods  of  Analysis,  and  Detection  of  Adulteration.  J. 
Am.  Chem.  Soc.,  26,  1523. 

LAXA  :  (Determination  of  Sucrose  and  Lactose  in  Milk  Chocolate). 
Z.  Nahr.  Genussm.,  7,  471.  t 

1905.  BAKER  and  DICK  :  The  Detection  and  Estimation  of  Small  Quantities 

of  Maltose  in  the  Presence  of  Dextrose.     Analyst,  30,  79. 

PFYL  and  LINNE  :  Hydrolysis  of  Sucrose,  Maltose,  Lactose  and  Ram- 
nose.  Z.  Nahr.  Genussm.,  10,  104. 

SAWYER  :  The  Commercial  Analysis  of  Cane  Molasses.  J.  Am.  Chem. 
Soc.,  27,  691. 

1906.  DAVOLL  :  An  Accurate  Commercial  Method  for  the  Analysis  of  Sugar 

Beets.     /.  Am.  Chem.  Soc.,  28,  1606. 
HORNE  :  Chemical  Control  of  Cane  Sugar  Factories.     School  of  Mines 

Quarterly,  27,  128. 
TOLMAN  and  SMITH  :  Estimation  of  Sugars  by  Means  of  the  Refrac- 

tometer.     J.  Am.  Chem.  Soc.,  28,  1476. 
WIN  TON  and  KREIDER:  Determination  of  the  "Lead  Number"  in 

Maple  Syrup  and  Maple  Sugar.     J.  Am.  Chem.  Soc.,  28,  1204. 

1907.  ANDRLIK  and  STANEK:  (Use  of  Hydrochloric  Acid  and  Urea  in  Direct 

Polarization  for  Clerget  Method).  Z.  Zuckerind.  JBdhmen,  31, 
417;  Chem.  Abs.,  1,  1912. 

BATES  and  BLAKE  :  Influence  of  Basic  Lead  Acetate  on  the  Rota- 
tion of  Sucrose  in  Water  Solution.  J.  Am.  Chem.  Soc.,  29,  286. 

HORNE  :  Dry  Lead  Clarification.     /.  Am.  Chem.  Soc.,  29,  926. 

KONIG  and  HORMANN  :  Separation  of  Carbohydrates  by  Pure  Yeasts. 
Z.  Nahr.  Genussm.,  13,  113. 

U.  S.  TREASURY,  DOCUMENT  No.  2470:  Regulations  Governing  the 
Weighing,  Taring,  Sampling,  Classification,  and  Polarization  of 
Imported  Sugars  and  Molasses. 

1908.  BROWNE  :  Chemical  Analysis  and  Composition  of  American  Honeys. 

Bui.  110,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 

BROWNE  and  HALLIGA'N  :  Report  on  Sugar  and  Molasses  Methods. 
U.  S.  Dept.  Agriculture,  Bur.  Chein.,  Bui.  116,  p.  68. 

LING  and  RENDLE  :  Determination  of  Sucrose  arid  Invert  Sugar  in 
Mixtures.  Analyst,  33,  167. 

SY:  Apparatus  for  Polarizing  at  87°.     J.  Am.  Chem.  Soc.,  30,  1790. 

U.  S.  DEPT.  AGRICULTURE,  Bur.  Chem.,  Bui.  107  (Revised)  :  Offi- 
cial and  Provisional  Methods  of  Analysis. 

1909.  BROWNE  :  Use  of  Temperature  Corrections  in  the  Polarization  of  Raw 

Sugars  and  other  Products  upon  Quartz  Wedge  Saccharimeters. 
J.  Ind.  Eng.  Chem.,  1,  567. 

Unification  of  Saccharimeter  Observations.     U.  S.  Dept.  Agr., 
Bur.  Chem.,  Bui.  122,  p.  221. 


SPECIAL   METHODS    OF   SUGAR   ANALYSIS  105 

Report  of  the  New  York  Sugar  Trade  Laboratory.     Z.  Ver. 

Zuckerind.,  59,  II,  297. 

HERZFELD  :  Estimation  of  Sugar  in  the  Sugar  Beet.  Z.  Ver.  Zuck- 
erind., 59,  627.  Chem.  Abs.,  3,  2249. 

HORNE  :  Clarification  with  Dry  Lead  Subacetate  in  the  Analysis  of 
Raw  Sugars.  Z.  Ver.  Zuckerind.,  59,  639  ;  Chem.  Abs.,  3,  2393. 

1910.  BAKER  and  HULTON  :  The  Estimation  of  Lactose  in  the  Presence  of 

the  Commonly  Occurring  Sugars.     Analyst,  35,  512. 
BROWNE  :  The  Normal  Weight  of  Dextrose.     J.  Ind.  Eng.  Chem.,  2, 

526. 
BROWNE  and  BRYAN  :  Lead  Clarification  in  Sugar  Analysis.     Z.  Ver. 

Zuckerind.,  59,  922;  Chem.  Abs.,  4,  258. 
HUDSON:  The   Quantitative  Determination  of    Cane  Sugar   by  the 

Use  of  Invertase.     J.  Ind.  Eng.  Chem.,  2,  143. 
LING,  EYNON,  and  LANE  :  Solution  Densities  of  Dextrose,  Levulose, 

and  Maltose.     J.  Soc.  Chem.  Ind.,  28,  730. 
REES  :  Optically  Active  Non-Sugars  of  the  Sugar  Beet;     J.  Ind.  Eng. 

Chem.,  2,  323. 
SPENCER  :  The  Milling  of  Cane  in  Sugar  Manufacture  Control.     J. 

Ind.  Eng.  Chem.,  2,  253. 
WITTE  :  Examination  of  Honey.     Z.  Nahr.  Genussm.,  21,  305. 

1911.  ANDRLIK  and  STANEK  :  Determination  of  Solids,  Coefficient  of  Purity, 

Loss  of  Polarization  during  Saturation,  and  Amount  of  Non -Sugar 
removed  during  Clarification  in  Diffusion  and  Saturated  Juices. 
Z.  Zuckerind.  Bohmen,  35,  257;  Chem.  Abs.,  5,  2751. 

BRYAN:  Analyses  of  Sugar  Beets  (with  Methods  and  Bibliography). 
U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  146. 

DUBOIS  :  (Determination  of  Sucrose  and  Lactose  in  Milk  Chocolate). 
U.  S.  Dept.  Agr.,  Bur.  Chem.,  Bui.  137,  p.  98. 

REISER:  Contribution  to  the  Chemistry  of  Honey,  with  Special 
Reference  to  Differences  from  Artificial  Wares.  Arb.  kais. 
Gesundh.,  30,  637;  Chem.  Abs.,  5,  538. 

PELLET  :  Determination  of  Sucrose  in  Cane  Molasses.  Intern.  Sugar 
J.,  13,  206 ;  Chem.  Abs.,  5,  2752. 

SCHONROCK:  Refractive  Index  of  Sugar  Solutions.  Z.  Ver.  Zuck- 
erind., 61,  421 ;  Chem.  Abs.,  5,  2575. 


CHAPTER  V 
Starch  and  Amylase 

THE   DETERMINATION   OF  STARCH 
METHOD  OF  DIRECT  ACID  HYDROLYSIS 

THIS  method  is  accurate  for  the  determination  of  starch  in 
such  samples  as  contain  no  other  substance  insoluble  in  water 
and  capable  of  yielding  reducing  sugar  on  heating  with  dilute 
acid.  It  is  now  well  known  that  such  interfering  substances, 
especially  the  pentosans,  are  generally  found  in  vegetable  tis- 
sues, and  that  the  results  obtained  by  direct  acid  hydrolysis 
are  usually  too  high.  This  is,  however,  the  method  which  has 
been  commonly  used  until  recently,  and  for  comparisons  with 
earlier  work  determinations  by  direct  hydrolysis  are  still  fre- 
quently required. 

Conversion  of  Starch  to  Dextrose  l 

Weigh  2  to  5  grams  and,  if  much  fat  is  present,  wash  with 
four  or  five  successive  portions  each  10  cc.  of  ether ;  reject  the 
filtrate,  allow  the  adhering  ether  to  evaporate  from  the  residue, 
and  wash  with  150  cc.  of  10  per  cent  alcohol  to  remove  soluble 
carbohydrates.  Wash  the  residue  into  a  250  cc.  flask  with 
200  cc.  of  water,  add  20  cc.  of  hydrochloric  acid  of  1.125  sp.  gr., 
and  heat  in  a  boiling  water  bath  with  a  reflex  condenser  for  two 
and  one  half  hours.  Cool  to  room  temperature,  nearly  neutralize 
with  sodium  hydroxide,  dilute  to  250  cc.,  filter,  and  determine 
dextrose  in  a  portion  of  the  filtrate,  using  either  Defren's 
method,  already  described,  or  Allihn's  method,  as  follows : 

1  Sachsse,  Chem.  CentrbL,  1877,  732  ;  Bui.  107,  Bur.  Chem.,  U.  S.  Dept.  Agri- 
culture. 

106 


STARCH   AND   AMYLASE  107 

Allihn's  Method  for  the  Determination  of  Dextrose1 

Reagents.  —  (1)  34.64  grams  of  crystallized  copper  sulphate 
dissolved  in  water  and  diluted  to  500  cc. 

(2)  173  grams  of  sodium  potassium  tartrate  and  125  grams 
of  potassium  hydroxide  dissolved  in  water  and  diluted  to 
500  cc. 

Determination.  —  Place  30  cc.  of  the  copper  solution,  30  cc.  of 
the  alkaline  tartrate  solution  and  60  cc.  of  water  in  a  beaker  or 
casserole  and  heat  to  boiling.  To  the  boiling  liquid  add  25  cc. 
of  the  dextrose  solution ;  note  the  time  at  which  the  mixture 
begins  to  boil  and  continue  the  boiling  for  exactly  two  minutes. 
Filter  at  once  and  obtain  the  weight  of  copper  in  the  precipitated 
cuprous  oxide  by  any  of  the  methods  suggested  in  connection 
with  Defren's  process  described  in  Chapter  III,  or  as  follows: 
Collect  the  cuprous  oxide  on  an  asbestos  filter  in  a  Gooch  cru- 
cible; wash  the  precipitate  (including  any  which  may  adhere 
to  the  beaker  and  which  need  not  be  transferred  to  the  filter) 
thoroughly  with  hot  water,  transfer  the  asbestos  and  adhering 
oxide  from  the  crucible  to  the  beaker.  Dissolve  the  oxide  still 
remaining  in  the  crucible  by  means  of  1  to  2  cc.  of  concentrated 
nitric  acid,  adding  the  acid  from  a  pipette  and  receiving  the 
solution  in  the  beaker  containing  the  asbestos  and  the  main 
part  of  the  precipitate.  Rinse  the  crucible  with  a  jet  of  water, 
allowing  the  rinsings  to  flow  into  the  beaker.  Heat  the  con- 
tents of  the  beaker  until  all  copper  is  in  solution;  filter,  wash 
thoroughly,  dilute  the  filtrate  to  100  to  150  cc.,  add  one  drop 
of  concentrated  sulphuric  acid  and  determine  copper  by  electrol- 
ysis. Find  the  corresponding  weight  of  dextrose  from  Allihn's 
table. 

Notes.  — The  conditions  described  must  be  observed  carefully. 
The  dextrose  solution  added  to  the  copper  reagent  must  be  free 
from  turbidity  and  only  faintly  acid.  The  rapid  addition  of 
this  cold  solution  stops  the  boiling  of  the  reagent,  and  it  is  well 
to  have  the  heat  so  regulated  that  the  mixture  will  boil  again 
in  about  two  minutes  ;  then,  after  exactly  two  minutes  of  actual 

1  J.  prakt.  Chem.,  1880,  22,  46 ;  Bui.  107,  loc.  cit. 


108 


METHODS   OF   ORGANIC   ANALYSIS 


TABLE  9.  —  ALLIHN'S  TABLE  FOR  THE  DETERMINATION  OF  DEXTROSE 


Milli- 
grams 
of 
copper 

Milli- 
grams 
of  dex- 
trose 

Milli- 
grams 
of 
copper 

Milli- 
grams 
of  dex- 
irose 

Milli- 
grams 
of 
copper 

Milli- 
grams 
of  dex- 
trose 

Milli- 
grams 
of 
copper 

Milli- 
grams 
of  dex- 
trose 

Milli- 
grams 
of 
copper 

Milli- 
grams 
of  dex- 
trose 

10 

6.1 

67 

34.3 

124 

63.1 

181 

92.6 

238 

122.8 

11 

6.6 

68 

34.8 

125 

63.7 

182 

93.1 

239 

123.4 

12 

7.1 

69 

35.3 

126 

64.2 

183 

93.7 

240 

123.9 

13 

7.6 

70 

35.8 

127 

64.7 

184 

94.2 

241 

124.4 

14 

8.1 

71 

36.3 

128 

65.2 

185 

94.7 

242 

125.0 

15 

8.6 

72 

36.8 

129 

65.7 

186 

95.2 

243 

125.5 

16 

9.0 

73 

37.3 

130 

66.2 

187 

95.7 

244 

126.0 

17 

9.5 

74 

37.8 

131 

66.7 

188 

96.3 

245 

126.6 

18 

10.0 

75 

38.3 

132 

67.2 

189 

96.8 

246 

127.1 

19 

10.5 

76 

38.8 

133 

67.7 

190 

97.3 

247 

127.6 

20 

11.0 

77 

39.3 

134 

68.2 

191 

97.8 

248 

128.1 

21 

11.5 

78 

39.8 

135 

68.8 

192 

98.4 

249 

128.7 

22 

12.0 

79 

40.3 

136 

69.3 

193 

98.9 

250 

129.2 

23 

12.5 

80 

408 

137 

69.8 

194 

99.4 

251 

129.7 

24 

13.0 

81 

41.3 

138 

70.3 

195 

100.0 

252 

130.3 

25 

13.5 

82 

41.8 

139 

70.8 

196 

100.5 

253 

130.8 

26 

14.0 

83 

42.3 

140 

71.3 

197 

101.0 

254 

131.4 

27 

14.5 

84 

42.8 

141 

71.8 

198 

101.5 

255 

131.9 

28 

15.0 

85 

43.4 

142 

72.3 

199 

102.0 

256 

132.4 

29 

15.5 

86 

43.9 

143 

72.9 

200 

102.6 

257 

133.0 

30 

16.0 

87 

44.4 

144 

73.4 

201 

103.1 

258 

133.5 

31 

16.5 

88 

44.9 

145 

73.9 

202 

103.7 

259 

134.1 

32 

17.0 

89 

45.4 

146 

74.4 

203 

104.2 

260 

134.6 

33 

17.5 

90 

45.9 

147 

74.9 

204 

104.7 

261 

135.1 

34 

18.0 

91 

46.4 

148 

75.5 

205 

105.3 

262 

135.7 

35 

18.5 

92 

46.9 

149 

76.0 

206 

105.8 

263 

136.2 

36 

18.9 

93 

47.4 

150 

76.5 

207 

106.3 

264 

136.8 

37 

19.4 

94 

47.9 

151 

77.0 

208 

106.8 

265 

137.3 

38 

19.9 

95 

48.4 

152 

77.5 

209 

107.4 

266 

137.8 

39 

20.4 

96 

48.9 

153 

78.1 

210 

107.9 

267 

138.4 

40 

20.9 

97 

49.4 

154 

78.6 

211 

108.4 

268 

138.9 

41 

21.4 

98 

49.9 

155 

79.1 

212 

109.0 

269 

139.5 

42 

21.9 

99 

50.4 

156 

79.6 

213 

109.5 

270 

140.0 

43 

22.4 

100 

50.9 

157 

80.1 

214 

110.0 

271 

140.6 

44 

22.9 

101 

51.4 

158 

80.7 

215 

110.6 

272 

141.1 

45 

23.4 

102 

51.9 

159 

81.2 

216 

111.1 

273 

141.7 

46 

23.9 

103 

52.4 

160 

81.7 

217 

111.6 

274 

142.2 

47 

24.4 

104 

52.9 

161 

82.2 

218 

112.1 

275 

142.8 

48 

24.9 

105 

53.5 

162 

82.7 

219 

112.7 

276 

143.3 

49 

25.4 

106 

54.0 

163 

83.3 

220 

113.2 

277 

143.9 

50 

25.9 

107 

54.5 

164 

83.8 

221 

113.7 

278 

144.4 

51 

26.4 

108 

55.0 

165 

84.3 

222 

114.3 

279 

145.0 

52 

26.9 

109 

55.5 

166 

84.8 

223 

114.8 

280 

145.5 

53 

27.4 

110 

56.0 

167 

85.3 

224 

115.3 

281 

146.1 

54 

27.9 

111 

56.5 

168 

85.9 

225 

115.9 

282 

146.6 

55 

28.4 

112 

57.0 

169 

86.4 

226 

116.4 

283 

147.2 

56 

28.8 

113 

57.5 

170 

86.9 

227 

116.9 

284 

147.7 

57 

29.3 

114 

58.0 

171 

87.4 

228 

117.4 

285 

148.3 

58 

29.8 

115 

58.6 

172 

87.9 

229 

118.0 

286 

148.8 

59 

30.3 

116 

59.1 

173 

88.5 

230 

118.5 

287 

149.4 

60 

30.8 

117 

59.6 

174 

89.0 

231 

119.0 

288 

149.9 

61 

31.3 

118 

60.1 

175 

89.5 

232 

119.6 

289 

150.5 

62 

31.8 

119 

60.6 

176 

90.0 

233 

120.1 

290 

151.0 

63 

32.3 

120 

61.1 

177 

90.5 

234 

120.7 

291 

151.6 

64 

32.8 

121 

61.6 

178 

91.1 

235 

121.2 

292 

152.1 

65 

33.3 

122 

62.1 

179 

91.6 

236 

121.7, 

293 

152.7 

66 

33.8 

123 

62.6 

180 

92.1 

237 

122.3 

294 

153.2 

STARCH   AND   AMYLASE 


109 


ALLIHN'S  TABLE  FOR  THE  DETERMINATION  OF  DEXTROSE — Continued 


Milli- 
grams 
of 
copper 

Milli- 
grams 
of  dex- 
trose 

Milli- 
grams 
of 
copper 

Milli- 
grams 
of  dex- 
trose 

Milli- 
grams 
of 
copper 

Milli- 
grams 
of  dex- 
trose' 

Milli- 
grams 
of 
copper 

Milli- 
grams 
of  dex- 
trose 

Milli- 
grams 
of 
copper 

Milli- 
grams 
of  dex- 
trose 

295 

153.8 

329 

172.5 

363 

191.7 

397 

211.2 

431 

231.0 

296 

154.3 

330 

173.1 

364 

192.3 

398 

211.7 

432 

231  .6 

297 

154.9 

331 

173.7 

365 

192.9 

399 

212.3 

433 

232.2 

298 

155.4 

332 

174.2 

366 

193.4 

400 

212.9 

434 

232.8 

299 

156.0 

333 

174.8 

367 

194.0 

401 

213.5 

435 

233.4 

300 

156.5 

334 

175.3 

368 

194.6 

402 

214.1 

436 

233.9 

301 

157.1 

335 

175.9 

369 

195.1 

403 

214.6 

437 

234.5 

302 

157.6 

336 

176.5 

370 

195.7 

404 

215.2 

438 

235.1 

303 

158.2 

337 

177.0 

371 

196.3 

405 

215.8 

439 

235.7 

304 

158.7 

338 

177.6 

372 

196.8 

406 

216.4 

440 

236.3 

305 

159.3 

339 

178.1 

373 

197.4 

407 

217.0 

441 

236.9 

306 

159.8 

340 

178.7 

374 

198.0 

408 

217.5 

442 

237.5 

307 

160.4 

341 

179.3 

375 

198.6 

409 

218.1 

443 

238.1 

308 

160.9 

342 

179.8 

376 

199.1 

410 

218.7 

444 

238.7 

309 

161.5 

343 

180.4 

377 

199.7 

411 

219.3 

445 

239.3 

310 

162.0 

344 

180.9 

378 

200.3 

412 

219.9 

446 

239.8 

311 

162.6 

345 

181.5 

379 

200.8 

413 

220.4 

447 

240.4 

312 

163.1 

346 

182.1 

380 

201.4 

414 

221.0 

448 

241.0 

313 

163.7 

347 

182.6 

381 

202.0 

415 

221.6 

449 

241.6 

314 

164.2 

348 

183.2 

382 

202.5 

416 

222.2 

450 

242.2 

315 

164.8 

349 

183.7 

383 

203.1 

417 

222.8 

451 

242.8 

316 

165.3 

350 

184.3 

384 

203.7 

418 

223.3 

452 

243.4 

317 

165.9 

351 

184.9 

385 

204.3 

419 

223.9 

453 

244.0 

318 

166.4 

352 

185.4 

386 

204.8 

420 

224.5 

454 

244.6 

319 

167.0 

353 

186.0 

387 

205.4 

421 

225.1 

455 

245.2 

320 

167.5 

354 

186.6 

388 

206.0 

422 

225.7 

456 

245.7 

321 

168.1 

355 

187.2 

389 

206.5 

423 

226.3 

457 

246.3 

322 

168.6 

356 

187.7 

390 

207.1 

424 

226.9 

458 

246.9 

323 

169.2 

357 

188.3 

391 

207.7 

425 

227.5 

459 

247.5 

324 

169.7 

358 

188.9 

392 

208.3 

426 

228.0 

460 

248.1 

325 

170.3 

359 

189.4 

393 

208.8 

427 

228.6 

461 

248.7 

326 

170.9 

360 

190  ro 

394 

209.4 

428 

229.2 

462 

249.3 

327 

171.4 

361 

190.6 

395 

210.0 

429 

229.8 

463 

249.9 

328 

172.0 

362 

191.1 

396 

210.6 

430 

230.4 

boiling,  remove  the  flame  and  filter  the  solution  at  once.  The 
cuprous  oxide  is  very  apt  to  run  through  the  filter.  To  prevent 
this,  after  making  the  asbestos  filter  as  usual,  pour  on  it  some 
very  fine  asbestos  suspended  in  water,  so  as  to  form  a  tight 
layer  on  the  top  of  the  felt.  On  the  assumption  that  the  starch 
is  quantitatively  hydrolyzed  to  dextrose,  the  weight  of  the  latter 
multiplied  by  0.9  gives  the  corresponding  weight  of  starch. 
For  detailed  studies  of  the  hydrolysis  of  starch  by  acids,  see 
the  papers  of  Rolfe l  and  of  Noyes.2  According  to  several 

1  J.  Am.  Chem.  Soc.,  1896,  18,  869  ;  1897,  19,  261;  1903,  25,  1003,  1015. 

2  Ibid.,  1904,  26,  266. 


110  METHODS    OF   ORGANIC    ANALYSIS 

investigators,1  the  weight  of  dextrose  should  be  multiplied 
by  a  higher  factor  (0.917  to  0.941)  rather  than  0.9,  to  find  the 
corresponding  weight  of  starch. 

METHOD  OF  DIGESTION  WITH  DIASTASE  OR  SALIVA 

If  starch  is  gelatinized  by  boiling  with  water  and  then  treated 
with  malt  diastase  or  saliva,  it  can  be  converted  into  maltose 
and  dextrin  and  these  separated  by  filtration  from  the  insoluble 
residue  containing  the  pentosans  and  other  substances  which 
cause  the  results  by  the  preceding  method  to  be  too  high. 

Determination.  —  Extract  2.5  to  5  grams  of  sample  with  five 
successive  portions  each  10  cc.  of  ether,  decanting  the  washings 
through  a  hardened  filter ;  wash  with  150  cc.  of  10  per  cent 
alcohol ; 2  transfer  the  residue  to  a  beaker  with  50  to  100  cc.  of 
water  ;  heat  gradually  to  boiling,  stirring  constantly  to  prevent 
bumping  or  the  formation  of  lumps.  Cool  to  55°  for  diastase, 
or  to  38°  for  saliva ;  add  the  solution  containing  the  enzyme 
and  keep  the  mixture  within  two  degrees  of  the  stated  tem- 
perature until  a  drop,  removed  and  tested  on  a  porcelain  plate, 
no  longer  shows  a  reaction  for  starch  on  mixing  with  a  drop  of 
dilute  solution  of  iodine  in  aqueous  potassium  iodide.  Now 
heat  the  solution  again  to  boiling  in  order  to  gelatinize  any 
starch  granules  which  may  remain ;  test  the  solution,  and  if 
starch  is  found,  cool  to  the  proper  temperature ;  add  more  of  the 
enzyme  and  digest  as  before.  Continue  this  treatment  until 
the  solution  gives  no  starch  reaction  after  boiling,  or  until 
a  careful  microscopic  examination  shows  that  the  insoluble 
residue  is  entirely  free  from  starch.  Dilute  to  250  cc.,  mix 
thoroughly  and  pour  on  a  dry  fluted  filter.  Transfer  150  cc.  of 
the  filtrate  to  a  250-cc.  flask  ;  add  15  cc.  of  hydrochloric  acid  of 
1.125  sp.  gr.,  attach  the  flask  to  a  reflux  condenser  and  heat  in 

1  Salomon:    Z.    anal.    Chem.,    1883,    22,   593.     Soxhlet:    Wochenschr.  fur 
Brauer.,    1885,   193.     Vaubel,  II,  455.     Sostegni :    Chem.  ZentrbL,   1887,  58, 
896.     Lintner  and  Dtill :  Z.  angew.  Chem.,  1891,  537.     Ost :   Chem.  Ztg.,  1895, 
19,  1502.     Rossing  :  Z.  offentl.  Chem.,  1904,  10,  61  ;  Abs.  J.  Chem.  Soc.,  1904, 
86,  ii,  298.    Noyes  :  J.  Am.  Chem.  Soc.,  1904,  26,  280. 

2  This  extraction  can  often  be  omitted,  since  for  many  purposes  it  is  unneces- 
sary to  distinguish  between  starch  and  soluble  carbohydrates. 


STARCH   AND   AMYLASE  111 

a  boiling  water  bath  for  two  and  one  half  hours,  or  boil  gently 
on  a  hot  plate  or  sand  bath  for  35  to  45  minutes.  Complete  the 
determination  as  described  in  the  preceding  method. 

The  determination  should  be  carried  through  without  inter- 
ruption. If  this  is  impossible,  care  must  be  taken  to  avoid 
alcoholic  or  lactic  fermentation.  After  the  digestion  with  the 
enzyme  is  finished,  but  not  before,  salicylic  acid  may  be  added 
as  a  preservative.  It  has  been  recommended  that  a  trace  of 
fluoride  be  added  at  the  start  to  retard  lactic  fermentation 
while  the  digestion  with  the  enzyme  is  taking  place. 

When  only  a  few  determinations  are  to  be  made,  freshly  col- 
lected saliva  can  conveniently  be  used,  as  this  is  free  from  car- 
bohydrate. If  commercial  diastase  or  an  infusion  of  malt 1  is 
used,  the  amount  added  must  be  noted  and  a  correction  applied 
for  the  carbohydrate  thus  introduced.  This  is  found  by  heating 
a  quantity  of  the  diastase  or  infusion  with  acid  and  determining 
the  resulting  dextrose  as  in  the  starch  determination. 

COMPARISON  OF  RESULTS 

The  diastase  method,  carefully  carried  out,  is  believed  to 
yield  practically  correct  results.  As  already  explained,  the 
results  obtained  by  direct  acid  hydrolysis  are  usually  higher 
owing  to  the  presence  of  other  substances  which  yield  reducing 
sugars.  A  comparison  of  the  results  of  the  two  methods  is  of 
considerable  interest,  both  because  many  of  the  recorded  de- 
terminations of  starch  were  made  by  the  acid  method  and 
because  the  determination  of  copper-reducing  substance  ob- 
tained by  direct  hydrolysis  is  sometimes  used  as  a  means  of 
detecting  adulterants  in  spices.  The  table  below  shows  the 
results  of  comparison  of  the  two  methods  on  a  variety  of  sub- 
stances. Many  of  the  results  of  Winton  and  associates  are 
averaged  from  the  comparative  examination  of  several  samples. 
The  other  results  were  obtained  by  the  writer,  only  one  sample 
of  each  kind  being  examined. 

1  An  active  malt  infusion  can  be  prepared  by  digesting  10  grams  of  fresh, 
finely-ground  malt,  overnight  at  room  temperature,  with  200  cc.  of  water  or  10 
per  cent  alcohol. 


112  METHODS   OF   ORGANIC   ANALYSIS 

TABLE  10.  —  RESULTS  BY  DIASTASE  AND  BY  DIRECT  ACID  HYDROLYSIS 


Substance 

Starch  indicated  by 

Substance 

Starch  indicated  by 

Diastase 
method 
Per  cent 

Acid 
method 
Per  cent 

Diastase 
method 
Per  cent 

Acid 
method 
Per  cent 

Air-dry  starch  .  .  . 
Wheat  flour  .  .  . 
Oatmeal  » 

82.49 

66.55 
56.23 
55.32 
20.97 
14.06 
8.07 
4.39 
4.14 
1.46 
0.96 

82.30 

68.35 
59.01 
58.63 
38.82 
21.15 
11.16 
22.69 
18.03 
20.51 
20.13 

White  pepper  l    .     . 
Long  pepper  l      .     . 
Black  pepper  x     .     . 
Mace  1   
Nutmeg  l    .     .     .     . 
Allspice  l    .     .     .     . 
Cloves  ^  

56.47 

39.55 
34.15 
27.87 
23.72 
3.04 
2.74 
2.30 
1.01 
1.01 
0.84 

59.17 

42.88 
38.63 
31.73 
25.56 
18.03 
8.99 
11.43 
8.47 
19.30 
22.72 

Graham  flour  .  .  . 
Wheat  bran  .  .  . 
Linseed  meal  1.  ..  . 
Cocoa  nibs  l  .  .  . 
Wheat  straw  .  .  . 
Cocoa  shells  l  .  .  . 
Buckwheat  hulls  l  . 
Corn  stover  .... 

Pepper  shells  !     .     . 
Cayenne  l  .     .     .     . 
Walnut  shells  l   .     . 
Almond  shells  *  .     . 

OTHER  METHODS 

For  the  determination  of  starch  in  a  particular  sort  of 
material,  it  is  sometimes  feasible  to  obtain  accurate  results  by 
methods  shorter  than  those  above  described.  Thus,  the  starch 
in  cereals  is  sometimes  determined  by  dissolving  in  acid  and 
taking  the  rotatory  power  of  the  solution,  and  the  starch  con- 
tent of  potatoes  may  be  estimated  approximately  from  their 
specific  gravity.  Papers  describing  these  methods  are  included 
among  the  references  at  the  end  of  this  chapter. 

Starchy  materials  are  sometimes  added  as  u  fillers  "  to  sausages 
and  other  forms  of  chopped  meat.  This  adulteration  is  easily 
detected  by  the  iodine  reaction,  which,  however,  must  be  care- 
fully interpreted  since  a  small  amount  of  starch  may  legitimately 
be  present  from  the  spices  added  in  the  manufacture.  The 
quantitative  determination  is  complicated  by  the  fact  that  meat 
appears  to  contain  some  substance  which  interferes  with  the 
separation  of  the  cuprous  oxide  reduced  in  applying  the  usual 

1  Results  by  Winton  and  associates,  compiled  from  Leach's  Food  Inspection 
and  Analysis. 


STARCH   AND   AMYLASE  113 

method.  Advantage  is  therefore  taken  of  the  insolubility  of 
starch  in  alcoholic,  and  its  solubility  in  aqueous,  potassium  hy- 
droxide. The  method  of  Mayrhofer  as  modified  by  Bigelow 
and  adopted  by  the  Association  of  Official  Agricultural  Chemists 
is  as  follows  : l 

Treat  from  10  to  20  grams  of  the  sample  under  examination 
(depending  upon  the  amount  of  starch  indicated  by  the  iodine 
reaction)  in  a  porcelain  dish  or  casserole  with  50  cc.  of  an  8  per 
cent  solution  of  potassium  hydroxide  and  heat  the  mixture  on 
the  water  bath  until  the  meat  is  entirely  dissolved.  Add  an 
equal  volume  of  95  per  cent  alcohol,  mix  thoroughly,  filter  the 
mixture  through  an  asbestos  filter,  and  wash  twice  with  a  hot  4 
per  cent  solution  of  potassium  hydroxide  in  50  per  cent  alcohol. 
Then  wash  with  50  per  cent  alcohol  until  a  small  portion  of  the 
filtrate  does  not  become  turbid  upon  the  addition  of  acid. 
Return  the  precipitate  and  filter  to  the  original  vessel  and  dis- 
solve the  precipitate  with  the  aid  of  heat  in  60  cc.  of  a  normal 
solution  of  potassium  hydroxide."  In  the  case  of  sausage,  with  a 
high  starch  content,  a  somewhat  larger  volume  of  alkali  may  be 
required.  Acidify  the  filtrate  strongly  with  acetic  acid,  dilute 
to  a  definite  volume,  mix  thoroughly  by  shaking,  filter  through 
a  fluted  paper,  and  precipitate  the  starch  from  an  aliquot  part 
of  the  filtrate  by  means  of  an  equal  volume  of  95  per  cent 
alcohol.  Transfer  the  precipitate  to  a  weighed  filter,  wash 
thoroughly  with  50  per  cent  alcohol,  with  absolute  alcohol,  and 
finally  with  ether,  dry  to  a  constant  weight  at  the  temperature 
of  boiling  water,  and  weigh. 

DIASTATIC   POWER  OF  AMYLASES 

Of  interest  in  connection  with  the  determination  of  starch 
are  the  methods  for  the  determination  of  the  activity  or  power 
of  the  starch-splitting  enzymes  (amylases)  or  of  substances  con- 
taining such  enzymes.  Such  methods  are  of  growing  impor- 
tance, not  only  in  scientific  investigations,  but  also  in  industrial 
analysis. 

*Bul.  107,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 
i 


114  METHODS    OF   ORGANIC    ANALYSIS 

Malt  extracts  are  being  purchased  by  bakers  on  the  basis  of 
diastatic  power,  and  the  diastatic  power  of  malt  is  important 
in  the  alcohol  industry  when  it  is  desired  to  convert  as  large  an 
amount  of  foreign  starch,  by  the  use  of  as  little  malt,  as  possible. 
In  the  past,  malt  has  been  so  freely  used  in  the  alcohol  industry 
in  America  that  its  diastatic  power  has  been  a  matter  of  com- 
parative indifference,  but  it  is  to  be  expected  that  in  the  pro- 
duction of  industrial  alcohol  more  economical  methods  will  be 
employed,  and  the  proportion  of  malt  used  be  governed  by  its 
diastatic  power.  Already,  efforts  are  being  made  to  develop 
cheaper  sources  of  amylase  for  industrial  use. 

In  the  field  of  food  and  drug  inspection,  there  is  also  a  demand 
for  methods  for  the  determination  of  diastatic  power  as  a  means 
of  measuring  the  activity  of  the  various  starch-digesting  prep- 
arations which  are  offered  in  the  food  and  drug  trade  as  aids  to 
digestion  or  as  means  of  predigesting  starchy  food. 

Many  methods  of  determining  diastatic  power  have  been  pro- 
posed, but  most  of  these  have  been  tested  for  only  a  single 
amylase  or  only  a  narrow  range  of  conditions.  There  are  two 
main  types  of  methods,  based  respectively  upon  the  measurement 
of  the  amount  of  reducing  sugar  produced  by  the  amylase  or 
the  measurement  of  the  conversion  of  a  given  amount  of  starch 
into  hydrolytic  products. 

Evidently  the  result  obtained  by  either  type  of  method  will 
depend  upon  the  conditions  under  which  it  is  applied,  and 
further  it  may  be  expressed  in  a  number  of  ways,  for  example, 
the  amount  of  change  produced  by  a  given  amount  of  enzyme 
in  a  given  time,  the  amount  of  enzyme  required  to  produce  a 
given  change  in  a  given  time,  or  the  time  required  for  a  given 
amount  of  enzyme  to  produce  a  given  change. 

Three  typical  methods  are  outlined  below.  Whatever  method 
be  used,  the  observant  worker  will  find  that  the  results  may  be 
influenced  by  many  conditions  which  cannot  be  entered  into 
fully  here.  The  chemist  who  is  not  already  familiar  with  the 
difficulties  of  work  of  this  nature  should  not  rely  upon  the 
outlines  here  given,  but  should  also  study  carefully  the  original 
papers  cited  at  the  end  of  the  chapter. 


STARCH  AND  AMYLASE  115 

LINTNER'S  METHOD  AND  SCALE* 

Make  a  paste  of  two  grams  of  purified,2  dry,  soluble  starch 
in  a  little  cold  neutral  distilled  water  and  pour  into  50  or  60  cc. 
of  boiling  water.  Boil  for  a  minute  or  two  after  the  starch  is 
dissolved,  then  cool  and  dilute  to  100  cc.  in  a  graduated  flask. 
Place  10  cc.  of  this  starch  solution  in  each  of  a  series  of  ten 
test  tubes  and  place  the  tubes  in  a  water  bath  kept  at  21°  C. 

Weigh  out  50  to  100  milligrams  of  diastase  and  after  first 
mixing  with  4  or  5  cc.  of  water  dilute  to  100  or  200  cc.  accord- 
ing to  the  strength  of  the  enzyme.  Then  add  to  each  of  the 
starch  tubes  known  quantities  of  enzyme  solution  in  increasing 
amounts  (or  if  the  diastatic  power  of  malt  is  to  be  determined, 
add  to  each  tube  a  different  amount  of  malt  extract  prepared 
as  below),  e.g..  0.1,  0.2,  0.3,  0.4,  0.5  cc.,  etc.  After  digest- 
ing for  one  hour  at  21°  C.,  stop  the  action  by  the  addition  of 
5  cc.  of  Fehling  solution  to  each  tube,  place  the  tubes  in  boil- 
ing water  for  10  minutes  and  then  by  means  of  the  potassium 
ferrocyanide  test  (or  one  of  the  other  tests  described  in  Chapter 
III)  examine  to  determine  the  first  tube  in  which  the  copper  is 
all  reduced,  i.e.  the  smallest  amount  of  enzyme  or  extract 
which  has  produced  sufficient  maltose  to  reduce  5  cc.  of  Fehling 
solution.  Lintner  prepared  a  sample  of  diastase  of  which 
0.12  mg.  produced  under  these  conditions  the  maltose  necessary 
to  reduce  the  5  cc.  of  Fehling  solution.  This  preparation  was 
rated  as  having  a  diastatic  power  of  100  and  the  diastatic  powers 
of  other  preparations  were  calculated  as  inversely  proportional 
to  the  amount  of  sample  required  to  produce  this  fixed  amount 
of  reducing  sugar.  Thus,  if  0.3  mg.  were  required 

0.3  :  0.12:  :   100  :  X 
^T=40, 

the  diastatic  power  of  the  preparation  tested  (^Lintner  Scale). 

In  applying  this  method  to  malt,  digest  25  grams  of  the  dried, 
ground  sample  with  500  cc.  of  water  and  use  portions  of  the 

1  J.  prakt.  Chem.,  [2],  34,  378. 

2  The  preparation  of  soluble  starch  for  this  purpose  is  discussed  fully  by  Ford : 
J.  Soc.  Chem.  Ind.,  23,  414. 


116  METHODS  OF  ORGANIC  ANALYSIS 

clear  filtered  extract,  as  described  for  the  diastatic  solution. 
Then  if  V  =  volume  of  extract,  required  to  give  complete  re- 
duction of  the  Fehling  solution, 

V  i  0.1  ::  100  :  diastatic  power  of  malt  (Lintner  Scale). 

If  in  such  a  test  cupric  copper  be  found  in  the  tube  represent- 
ing 0.2  cc.  and  not  in  that  which  had  0.3  cc.  of  the  extract,  it 
is  evident  that  the  true  value  of  V  lies  either  at  0.8  cc.  or 
between  0.2  cc.  and  0.3  cc.  and  can  be  found  accurately  only 
by  repeating  the  test  with  small  increments  of  volume  between 
these  limits. 

Note  that  the  values  on  Lintner's  scale  for  malt  are  about 
40  times  as  high  as  on  his  scale  for  enzyme  preparations. 

WOHLGEMUTH'S  METHOD  AND  SCALE1 

Place  in  each  of  a  series  of  test  tubes  diminishing  amounts 
of  saliva  or  enzyme  solution  to  be  tested.  Introduce  into  each 
tube  5  cc.  of  a  1  per  cent  solution  of  soluble  starch  and  place 
each  tube  immediately  in  a  bath  of  ice  water.  When  all  are 
prepared,  transfer  the  tubes  to  a  water  bath  at  40°  for  30  minutes. 

At  the  end  of  this  time  transfer  again  immediately  to  the  ice 
water  bath  to  stop  the  action. 

.  Dilute  the  contents  of  each  tube  to  within  -|  inch  from  the 
top  with  water.  Add  one  drop  of  a  tenth-normal  solution  of 
iodine  and  shake  well.  One  usually  obtains  colors  from  blue 
through  violet,  red,  and  yellow  to  colorless  indicative  of  starch, 
erythrodextrin,  achroodextrin,  etc.  Taking  the  tube  where 
the  blue  or  violet  has  entirely  disappeared,  giving  place  either 
to  red  or  orange-red  color  as  judged  by  Mulliken's  Color 
Standard  Sheet  C,  note  the  amount  of  enzyme  solution  in 
this  tube  and  calculate  the  power  of  the  enzyme  as  the  number 
of  cubic  centimeters  of  1  per  cent  starch  solution  which  is 
digested  to  this  stage  in  a  given  time  by  1  cc.  of  enzyme  solution. 

Thus  if  0.2  gram  (or  cubic  centimeter)  of  the  substance 
tested  completely  digests  5  cc.  of  starch  solution  in  30  minutes 

1  Biochem.  Zeitschrift,  9,  1. 


STARCH   AND   AMYLASE  117 

at  40°,  then  1  gram  or  1  cc.  of  that  solution  will  be  able  to  digest 
250  cc.  of  1  per  cent  starch  solution,  or  its  power  is  then  stated  : 

D$=  250  (WoUgemuih  Scale). 

Here  the  time  of  reaction  is  brought  into  the  final  expression 
of  diastatic  power  so  that  the  method  may  be  applied  to  sub- 
stances whose  power  is  very  slight  by  allowing  them  to  act  a 
long  time. 

Wohlgemuth  has  used  the  method  principally  in  testing 
physiological  fluids,  in  which  case  the  results  are  expressions 
of  relative  volumes.  When  the  substance  tested  is  a  solid,  the 
results  are  calculated  in  the  same  way,  cubic  centimeter  and 
gram  being  considered  practically  interchangeable. 

NEW  METHOD  AND  SCALE  * 

Prepare  400  cc.  of  2  per  cent  soluble  starch  solution. 
Weigh  a  portion  (depending  upon  its  activity)  of  the  material 
to  be  tested,  dissolve,  and  dilute  to  volume  (Note  1).  By 
means  of  a  1  cc.  Mohr's  pipette  accurately  calibrated  in  hun- 
dredths,  measure  into  four  200  -cc.  Erlenmeyer  flasks  such 
volumes  of  the  enzyme  solution  as  will  contain  suitable  quan- 
tities of  .amylase  (Note  2).  Then,  noting  accurately  the  time 
at  which  the  starch  and  enzyme  solutions  are  mixed,  pour  into 
each  flask  100  cc.  of  the  starch  solution  which  has  been  pre- 
viously warmed  to  40°  (Note  3).  Place  the  flasks  immedi- 
ately in  a  water  or  air  bath,  the  temperature  of  which  is  main- 
tained at  exactly  40°  throughout  the  entire  time  of  reaction 
and  allow  to  digest  for  30  minutes  (Note  4).  At  the  expira- 
tion of  the  thirty  minutes  stop  the  action  immediately  by 
adding  to  each  flask  50  cc.  of  Fehling  solution  (Note  5),  and 
immersing  the  flasks  in  a  large  bath  of  boiling  water  for  15 
minutes.  See  that  the  water  in  the  bath  is  kept  boiling  and 
that  it  stands  above  the  level  of  the  contents  of  any  of  the 
flasks.2  At  the  end  of  this  heating  filter  quickty  on  weighed 

1  Sherman,  Kendall  and  Clark:  J.  Am.  Chem.  Soc.,  32,  1082. 

2  The  flasks  may  be  weighted  with  lead  rings  to  prevent  upsetting. 


118 


METHODS   OF   ORGANIC    ANALYSIS 


Gooch  crucibles  having  thick  felts  of  specially  prepared  asbes- 
tos ;  wash  the  cuprous  oxide  thoroughly  with  hot  water,  then 
with  alcohol  and  ether;  dry  for  at  least  20  minutes  in  a  boiling 
water  oven  and  weigh.  Use  for  the  following  calculation  that 
one  of  the  four  determinations  which  shows  the  highest  weight 
of  cuprous  oxide  not  exceeding  300  nig.  when  corrected  for 
the  reducing  power  of  the  soluble  starch. 

Correct  the  weight  of  cuprous  oxide  found  for  the  reducing 
power  of  the  soluble  starch  by  subtracting  from  it  the  weight 
obtained  in  a  "  blank "  test  in  which  the  starch  solution  is 
treated  directly  with  the  Fehling  reagent,  heated,  etc.,  exactly 
as  in  a  regular  determination. 

From  this  corrected  weight  of  cuprous  oxide  find  the  corre- 
sponding value  of  K  in  the  following  table,  and  divide  this 
value  for  K  by  the  weight  in  milligrams  of  the  substance 
under  examination  (Note  6). 


TABLE  11.  —  VALUES  FOR  K  FROM  CUPROUS  OXIDE  FOUND 


Cuprous 
oxide 
Mgms. 

K 

Cuprous 
oxide 
Mgms. 

K 

Cuprous 
oxide 
Mgms. 

K 

Cuprous 
oxide 
Mgms. 

K 

30 

9.1 

100 

31.2 

170 

54.1 

240 

78.3 

40 

12.2 

110 

34.4 

180 

57.5 

250 

81.8 

50 

15.3 

120 

37.6 

190 

60.9 

260 

85.4 

60 

18.4 

130 

40.9 

200 

64.3 

270 

89.0 

70 

21.6 

140 

44.2 

210 

67.8 

280 

92.6 

80 

24.8 

150 

47.5 

220 

71.3 

290 

96.3 

90 

28.0 

160 

50.8 

230 

74.8 

300 

100.0 

Example.  —  Suppose  a  flask  which  had  received  a  solution 
corresponding  to  1.2  mgm.  of  substance  shows  284.2  mgm. 
cuprous  oxide  and  the  "blank"  for  the  soluble  starch  is  60.2 
mgm.  Then  find  the  value  of  K  corresponding  to  284.2  — 
60.2  or  224  mgm.  cuprous  oxide.  This  is  72.7,  which  when 
divided  by  1.2  (the  weight  of  substance  acting)  gives  a  diet- 
static  power  of  66  (new  scale). 


STAKCH   AND   AMYLASE  119 

Note  1. — In  making  the  solutions  of  starch  and  of  enzyme 
the  purest  obtainable  water  should  be  used.  To  this. water 
should  be  added  the  necessary  "  activating  agents  "  to  enable 
the  enzyme  to  exert  its  normal  action.  In  testing  pancreatic 
amylase  300  mg.  sodium  chloride  and  7  cc.  fiftieth-molar  di- 
sodium  phosphate  are  added  to  100  cc.  of  water  used  as  solvent 
both  in  making  up  the  starch  solution  and  for  dissolving  the 
enzyme. 

Note  2.  —  The  amount  of  sample  required  will  vary  greatly 
with  the  nature  of  the  material.  In  testing  malts  a  volume  of 
extract  corresponding  to  several  milligrams  of  sample  may  be 
required;  in  testing  commercial  diastatic  preparations,  from  a 
fraction  of  a  milligram  to  a  few  milligrams ;  in  testing  highly 
purified  preparations  a  volume  of  solution  corresponding  to 
only  a  few  hundredths  of  a  milligram  of  the  substance  may  be 
required. 

Note  3.  — A  temperature  of  40°  C.  is  suitable  for  enzymes  of 
animal  origin,  and  as  those  of  vegetable  origin  have  heretofore 
been  tested  at  various  temperatures  from  21°  to  60°  C.  it  seems 
very  desirable  that  a  uniform  temperature  of  40°  be  adopted  for 
all  testing  of  amylases,  notwithstanding  the  fact  that  they  will 
usually  be  found  to  work  more  rapidly  at  a  higher  temperature. 
In  general,  results  obtained  by  different  observers  working  at 
different  temperatures  cannot  properly  be  compared. 

Note  4. — The  regulation  of  temperature  is  very  important, 
as  a  difference  of  1°  C.  (throughout  the  time  of  digestion)  under 
the  optimum  conditions  here  described  for  pancreatic  amylase 
affects  the  result  about  10  per  cent.  It  is  well  to  stand  the 
flasks  in  a  water  bath  containing  a  thermometer  and  place  the 
whole  in  an  air  bath.  The  importance  of  accurate  regulation  of 
the  time  is  obvious.  Should  the  time  of  action  of  the  enzyme 
be  other  than  30  minutes,  the  result  need  not  be  rejected,  but 
the  value  found  for  K  below  must  be  multiplied  by  30  and 
divided  by  the  actual  time  in  minutes,  before  dividing  by  the 
weight  of  enzyme  to  find  the  diastatic  power. 

Note  5.  —  Fehling  solution  as  described  under  Fehling's  or 
Defren's  method  in  Chapter  III  is  used.  The  addition  of  this 


120  METHODS   OF   ORGANIC   ANALYSIS 

to  each  solution  should  be  accurately  timed  so  that  the  time  of 
action  of  the  enzyme  on  the  starch  shall  be  exactly  known  in 
each  case. 

Note  6.  —  As  is  usual  in  such  cases  the  rate  of  amylolytic 
action  decreases  slightly  as  the  conversion  of  starch  into  maltose 
proceeds,  so  that  weights  of  maltose  or  of  cuprous  oxide  cannot 
be  taken  as  showing  directly  the  true  diastatic  power,  but  if  the 
velocity  curve  of  the  amylolytic  action  be  plotted  with  time  as 
abscissas  and  yield  of  reducing  sugar  as  ordinates  a  scale  may 
be  established  which  will  permit  of  an  expression  of  true  dia- 
static power  based  upon  the  weight  of  cuprous  oxide  obtained, 
as  above  described.  If  300  mg.  of  cuprous  oxide  be  taken  as 
100  on  such  a  scale,  the  value  on  this  scale  of  any  lesser  weight 
of  cuprous  oxide  is  obtained  by  expressing  the  abscissa  corre- 
sponding to  such  weight  as  percentage  of  the  abscissa  for  300 
mgm.  cuprous  oxide.  The  values  thus  obtained  are  given 
as  "values  for  JT"  in  the  table. 

UNIFICATION  OF  METHODS  FOR  DETERMINING  AND 
EXPRESSING  DIASTATIC  POWER 

The  data  at  present  available  are  not  sufficient  to  warrant  the 
proposal  of  any  one  method  as  being  the  most  accurate  and  the 
most  logical  for  all  cases.  It  remains  to  be  determined  whether 
the  power  of  forming  reducing  sugars  (saccharifying  power) 
and  the  power  of  converting  starch  into  products  which  do  not 
give  a  blue  color  with  iodine  ("  liquefying  "  power)  run  parallel 
as  between  amylases  of  different  origins.  If  not,  it  may  well  be 
that  the  use  for  which  the  amylase  is  intended  will  influence 
the  choice  of  method.  When  the  conditions  governing  the 
saccharifying  power  of  the  different  amylases,  and  the  products 
of  their  action,  have  been  more  fully  determined,  it  is  hoped  that 
a  simple  mode  of  expressing  diastatic  power  in  terms  of  a  ratio 
of  the  weight  of  active  substance  to  starch  converted  or  to  re- 
ducing sugar  formed  under  standard  conditions  may  be  found 
feasible  for  general  use. 

In  the  meantime  if  those  having  occasion  to  determine  and 


STARCH   AND   AMYLASE  121 

record  diastatic  powers  will  use  one  of  the  methods  above  de- 
scribed, the  comparison  of  results  obtained  by  different  workers 
will  be  greatly  facilitated  and  all  results  will  thereby  gain  in 
value. 

REFERENCES 


ABDERHALDEN  :  Biochemisches  Arbeitsmethoden. 

ALLEN  :   Commercial  Organic  Analysis. 

BROWN  :    Laboratory  Studies  for  Brewing  Students. 

EFFRONT  :   Enzymes  and  their  Applications. 

LUNGE  :   Chemisch-technisch  Untersuchungsmethoden. 

OPPENHEIMER  :   Die  Fermente  und  ihre  Wirkungen. 

POST  :   Chemisch-technische  Analyse. 

VAUBEL  :   Quantitative  Bestimmung  Organischer  Verbindungen. 

WILEY  :   Agricultural  Analysis. 

II 

(On  the  Determination  of  Starch) 

1888.  WINTON:   J.  Anal.  AppL  Chem.,  2,  153. 

1895.  OST  :    Chem.  Ztg.,  19,  1501. 

1896.  SHERMAN:    School  of  Mines  Quarterly,  17,  356. 
1898.  WILEY  and  KRUG  :   J.  Am.  Chem.  Soc.,  20,  253,  266. 
1904.  WITTE  :    Z.  Nahr.  Genussm.,  7,  65. 

1907.  LINTNER  :   Z.  Nahr.  Genussm.,  14,  205,  and  Z.  Ges.  Brauw.,  30,  109. 
PAROW  and  NEUMANN  :   Z.  Spiritusind.,  30,  561. 

1908.  DUBOIS:    (Cocoa  products).     U.  S.  Dept.  Agriculture,  Bur.  Chem., 

Bui.  122,  p.  214. 
HEIDE:    (Potatoes).     Chem.  Ztg.,  31,  398. 

1909.  THORNE  and  JEFFERS  :   J.  Soc.  Chem.  Ind.,  28,  731. 

(On  Amylases  and  the  Determination  of  Diastatic  Power) 

1880.  KJELDAHL  :    (First  systematic  study  of  method).     Dingler's polyt.  J., 

235,  379,  452. 
1886.   LINTNER  :    (Preparation  of  amylase  and  method  for  diastatic  power). 

/.  prate.  Chem.,  [2],  34,  378. 

1895-96.    OSBORNE  :    (Malt  Amylase).   /.  Am.  Chem.  Soc.,  17,  587;  18,  536. 
1896.   SYKES  and  MITCHELL  :    (Method).     A nalyst,  21,  122. 
1898.   FRANCIS:    (Method).     Bulletin  of  Pharmacy,  12,  52. 

TAKAMINE  :    (Method).     /.  Soc.  Chem.  2nd.,  17,  118,  437. 


122  METHODS  OF  ORGANIC  ANALYSIS 

1904.   FORD:    (Details  of  method  and  preparation  of  soluble  starch).     «/. 
Soc.  Chem.  Ind.,  23,  414. 

1907.  WOHLGEMUTH  :    (Method).     Biochem.  Z.,  9,  1. 

1908.  JOHNSON:    (Method).     J.  Am.  Chem.  Soc.,  30,  798. 

JONES:    (Discussion   of  Lintner  Method).     /.   Inst.  Brew.,  14,13; 

Chem.  Abs.,  2,  1592. 
LINTNER  and  WIRTH  :    (Method).     Z.  ges.  Brauw.,  31,421;  Chem. 

Abs.,  2,  3257. 

1910.  SHERMAN,  KENDALL,  and  CLARK  :  (Methods).     J.  Am.   Chem.  Soc., 

32,  1073. 

KENDALL  and  SHERMAN  :    (Pancreatic  Amylase).     J.  Am.  Chem.  Soc., 
32,  1087. 

1911.  SHERMAN  and  SCHLESINGER  :    (Pancreatic  Amylase).    /.  Am.  Chem. 

Soc.,  33, 1195. 


CHAPTER  VI 
Vinegar  and  Acetate 

VINEGAR 

THE  principal  varieties  of  vinegar  have  been  defined  and 
standardized  by  the  U.  S.  Department  of  Agriculture  as 
follows : 

Vinegar,  cider  vinegar,  apple  vinegar,  is  the  product  made  by 
the  alcoholic  and  subsequent  acetous  fermentations  of  the  juice 
of  apples,  is  levorotatory,  and  contains  not  less  than  4  grams  of 
acetic  acid,  not  less  than  1.6  grams  of  apple  solids,  of  which  not 
more  than  50  per  cent  are  reducing  sugars,  and  not  less  than 
0.25  gram  of  apple  ash  in  100  cc.  (20°  C.);  and  the  water- 
soluble  ash  from  100  cc.  (20°  C.)  of  the  vinegar  contains  not 
less  than  10  ing.  of  phosphoric  acid  (P2O5),  and  requires 
not  less  than  30  cc.  of  decinormal  acid  to  neutralize  its 
alkalinity. 

Wine  vinegar,  grape  vinegar,  is  the  product  made  by  the 
alcoholic  and  subsequent  acetous  fermentations  of  the  juice  of 
grapes,  and  contains  in  100  cc.  (20°  C.)  not  less  than  4  grams 
of  acetic  acid,  not  less  than  1.0  gram  of  grape  solids,  and  not 
less  than  0.13  gram  of  grape  ash. 

Malt  vinegar  is  the  product  made  by  the  alcoholic  and  subse- 
quent acetous  fermentations,  without  distillation,  of  an  infusion 
of  barley  malt,  or  cereals  whose  starch  has  been  converted  by 
malt,  is  dextrorotatory,  and  contains  in  100  cc.  (20°  C.)  not  less 
than  4  grams  of  acetic  acid,  not  less  than  2  grams  of  solids,  and 
not  less  than  0.2  gram  of  ash  ;  and  the  water  soluble  ash  from 
100  cc.  (20°  C.)  of  the  vinegar  contains  not  less  than  9  mg. 

123 


124  METHODS  OF  ORGANIC  ANALYSIS 

of  phosphoric  acid  (P2O5),  and  requires  not  less  than  4  cc.  of 
decinormal  acid  to  neutralize  its  alkalinity. 

Sugar  vinegar  is  the  product  made  by  the  alcoholic  and  sub- 
sequent acetous  fermentations  of  solutions  of  sugar,  syrup, 
molasses,  or  refiner's  syrup,  and  contains  in  100  cc.  (20°  C.) 
not  less  than  4  grams  of  acetic  acid. 

G-lucose  vinegar  is  the  product  made  by  the  alcoholic  and  sub- 
sequent acetous  fermentations  of  solutions  of  starch  sugar  or 
glucose,  is  dextrorotatory,  and  contains  in  100  cc.  (20°  C.)  not 
less  than  4  grams  of  acetic  acid. 

Spirit  vinegar,  distilled  vinegar,  grain  vinegar,  is  the  product 
made  by  the  acetous  fermentation  of  dilute  distilled  alcohol  and 
contains  in  100  cc.  (20°  C.)  not  less  than  4  grams  of  acetic  acid. 

Vinegar  of  any  of  these  varieties  may  contain  as  an  adulterant, 
or  substitute,  acetic  acid  made  by  refining  the  product  of  the  dry 
distillation  of  wood  and  which  is  sometimes  known  as  wood 
vinegar.  This  wood  vinegar  or  pyroligneous  acid  if  not  well 
refined  may  be  recognized  by  the  tarry  taste  and  odor  and 
identified  chemically  by  a  color  reaction  for  furfural  (see  quali- 
tative test  for  pentoses,  Chapter  III).  When  well  refined,  the 
dilute  solution  of  commercial  acetic  acid  cannot  be  recognized 
in  vinegar  by  any  characteristic  of  its  own,  but  only  by  its 
effect  in  lowering  the  amounts  of  solids,  ash,  etc.,  which  char- 
acterize the  pure  vinegars  of  the  different  varieties. 


DETERMINATION  OF  SOURCE 

The  principal  vinegar  of  the  United  States  is  cider  vinegar, 
although  considerable  quantities  of  malt  vinegar  are  also  used. 
The  substitutes  are  made  mainly  of  spirit  vinegar,  sugarhouse 
or  glucose  wastes,  and  wood  vinegar  from  acetate  of  lime. 
Cheap  apple  jelly  is  sometimes  added  to  the  substitutes  to  give 
them  the  color,  flavor,  and  body  of  cider  vinegar. 

Cider  vinegar  contains  from  1.2  to  8  per  cent  of  solids, 
average  about  2.5  per  cent ;  malt  vinegar  1.75  to  6,  average 
about  3  per  cent ;  spirit  vinegar  rarely  over  0.75  per  cent, 
average  about  0.30  per  cent. 


VINEGAR   AND   ACETATE  125 

The  total  ash  in  spirit  and  wood  vinegars  rarely  exceeds  0.1 
per  cent.  In  fruit  and  malt  vinegars  it  rarely  falls  below  0.2, 
cider  vinegar  averaging  about  0.35  per  cent. 

The  alkalinity  of  the  ash,  expressed  as  above,  is,  according 
to  Frear,  for  cider  vinegar  26  to  65,  average  39 ;  for  malt 
vinegar,  5.5  ;  for  spirit  vinegar,  1.1. 

The  flame  reaction  of  the  solids  is  said  to  be  always  that  of. 
potash  in  the  case  of  cider  vinegar,  while  spirit,  sugar,  and 
glucose  vinegars  and  any  which  have  been  artificially  colored 
show  the  sodium  flame  (Davenport-Frear). 

The  optical  activity  of  vinegar  often  indicates  the  source. 
Pure  cider  vinegar,  after  clarification  with  basic  lead  acetate, 
is  levorotatory,  a  200-mm.  tube  giving  usually  a  reading  of 
—  0.5°  to  —1.4°  on  the  Ventzke  scale.  According  to  Browne, 
levulose  is  the  only  sugar  present  in  properly  fermented  cider 
vinegar,  the  sucrose  and  dextrose  having  both  disappeared  in 
the  alcoholic  fermentation.  Wine  vinegar  is  also  slightly 
levorotatory.  Vinegar  from  sugarhouse  wastes  is  dextro- 
rotatory before,  and  levorotatory  after,  hydrolysis.  Glucose 
vinegar  shows  dextrorotation  both  before  and  after  hydrolysis. 
Artificial  vinegars  can,  of  course,  be  made  levorotatory  by  the 
addition  of  apple  juice  or  cider. 

It  has  been  supposed  that  the  presence  of  malic  acid  should 
be  one  of  the  characteristics  of  genuine  cider  vinegar,  but  the 
work  of  Van  Slyke  and  others  has  shown  that  malic  acid  of 
apple  juice  is  largely,  if  not  wholly,  destroyed  during  the 
alcoholic  and  acetous  fermentations,  so  that  tests  for  or  determi- 
nations of  malic  acid  are  of  little  if  any  value  in  the  examina- 
tion of  vinegar  as  to  its  source. 

Balcom  finds  that  determination  of  the  nonsugar  solids  by 
subtracting  the  total  sugar  from  the  total  solids  is  an  im- 
portant aid  in  the  judgment  of  vinegars.  Table  12  and  the 
explanations  which  follow  are  based  on  Balcom's  report  pub- 
lished in  1910.1 

1  U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  132. 


126 


METHODS    OF   ORGANIC   ANALYSIS 


TABLE  12.  — COMPARISON  OF  ANALYSES  OF  VINEGARS  OF  KNOWN  CHAR- 
ACTERS WITH  COMMERCIAL  SAMPLES  AND  WITH  AVERAGE  DATA 
(Balcom). 

[Grams  per  100  cc.] 


Total 
acid 

Total 

Non- 

Reduc- 
ing 

Total 

Alka- 
linity 
of 

Ash 
in 

Phosphoric  acid 
(P.O.) 

Polari- 

No.] 

as 
acetic 

solids 

sugar 
solids 

sugars 
in 
solids 

ash 

water- 
soluble 
ash 

non- 
sugar 
solids 

zation 
(direct) 

Solu- 
ble 

Insolu- 
ble 

Total 

Per  ct. 

cc. 

Per  ct. 

V 

!4.94 

2.54 

1.90 

19.6 

0.367 

35.7 

18.8 

17.3 

12.0 

29.3 

—  1.46 

11    .     . 

7.96 

4.52 

2.89 

45.0 

0.52 

56.0 

26.5 

39.9 

32.0 

64.2 

-3.6 

3.29 

1.37 

1.26 

5.6 

0.20 

21.5 

11.2 

6.7 

4.3 

15.1 

—  0.2 

2      .     . 

4.65 

2.40 

1.51 

37.2 

0.32 

31.9 

21.2 

12.4 

10.5 

22.9 

-1.3 

3      .    . 

4.31 

0.18 

0.16 

11.1 

0.016 

1.5 

10.0 

0.2 

1.5 

1.7 

+  0.6 

4      .     . 

4.51 

1.27 

0.80 

37.0 

0.20 

17.5 

25.0 

5.5 

7.4 

12.9 

-0.3 

5      .     . 

4.72 

2.15 

1.05 

51.2 

0.28 

37.0 

26.7 

8.7 

10.4 

19.1 



6      .     . 

4.46 

2.11 

0.91 

56.9 

0.29 

33.0 

31.9 

11.5 

10.8 

22.3 

—  1.0 

7      .     . 

4.66 

2.09 

1.73 

17.0 

0.56 

11.1 

32.4 

1.8 

15.2 

17.0 

±0.0 

No.  1  is  the  normal  basis  for  comparison  established  by  the 
compilation  of  about  100  analyses  of  cider  vinegars  of  known 
purity.  These  analyses  were  made  by  different  chemists  and 
represent  vinegars  from  different  parts  of  the  country,  al- 
though the  greater  number  were  of  vinegars  made  from  apples 
grown  in  New  England. 

No.  2  was  a  blend  of  about  50  commercial  samples  which 
from  analysis  were  passed  as  pure,  but  some  of  which  were 
regarded  as  not  above  suspicion. 

No.  3  was  an  uncolored  spirit  vinegar.  (Commercial  spirit 
vinegars  are  frequently  colored  with  caramel.) 

No.  4  was  a  mixture  of  equal  volumes  of  Nos.  2  and  3. 
Note  the  differences  in  analytical  data  between  this  sample  and 
No.  2  or  No.  1. 

No.  5  was  a  known  mixture  of  cider  vinegar,  spirit  vinegar, 
and  boiled  cider,  the  latter  bringing  up  the  total  solids,  etc. 
Note  the  low  value  of  the  nonsugar  solids  considered  in  con- 
nection with  the  abnormally  high  percentage  of  sugar  in  the 
total  solids. 

1  Average,  maximum,  and  mininum  data  on  about  100  vinegar  samples. 


VINEGAR   AND   ACETATE  127 

No.  6  was  a  commercial  sample  whose  analysis,  as  will  be 
seen,  shows  it  to  have  been  of  the  same  character  as  No.  5. 
The  analysis  of  No.  6  is  given  by  Balcom  as  typical  of  a  large 
number  of  vinegars  found  on  the  market  at  the  present  time 
and  which  are  regarded  as  mixtures  of  cider  vinegar  and 
dilute  acetic  acid,  the  latter,  perhaps,  in  the  form  of  spirit 
vinegar,  to  which  has  been  added  some  foreign  material  high 
in  sugar.  Whether  this  material  is  unfermented  apple  juice, 
boiled  cider,  apple  jelly,  or  some  other  material  is  difficult,  if 
not  impossible,  to  determine  with  certainty.  In  most  cases  it 
is  believed  to  be  either  unfermented  apple  juice  or  boiled  cider. 

No.  7  is  a  sugar  vinegar  made  from  New  Orleans  molasses 
and  is  supposed  to  be  fairly  representative  of  this  kind  of 
vinegar  which  is  being  introduced  to  an  increasing  extent 
to  take  the  place  of  the  cheap  artificially  colored  spirit  vinegar. 
It  will  be  noted  that  the  analytical  data  of  the  sugar  vinegar 
show  considerable  resemblance  to  that  of  a  cider  vinegar,  the 
most  noticeable  differences  being  a  relatively  higher  ash  or 
lower  alkalinity  of  the  water-soluble  ash,  low  water-soluble 
phosphoric  acid,  and  usually,  though  not  necessarily,  a  dif- 
ference in  behavior  toward  polarized  light.  The  latter  prop- 
erty will,  of  course,  depend  upon  the  nature  of  the  residual 
sugar,  as  determined  by  the  composition  of  the  sugar  syrup  or 
molasses  fermented. 

METHODS  OF  ANALYSIS 

A  thorough  examination  of  vinegar  should  include  determina- 
tions of  specific  gravity,  total  solids  or  extract,  total  and 
soluble  ash,  alkalinity  and  phosphoric  acid  of  the  ash,  sugars, 
polarization,  total  and  volatile  acidity,  and  alcohol.  Free 
mineral  acids  should  be  determined  if  present. 

Methods  for  all  of  these  determinations  have  been  adopted  by 
the  Association  of  Official  Agricultural  Chemists.  The  follow- 
ing directions  for  the  more  important  determinations  are  in 
accordance  with  the  official  methods.  It  is  not  necessary  to 
repeat  here  the  methods  for  alcohol,  sugars,  and  for  polari- 
zation. 


128  METHODS   OF   ORGANIC    ANALYSIS 

Total  Solids  or  Extract 

Evaporate  10  cc.  nearly  to  dry  ness  in  a  weighed  platinum 
dish  of  50  mm.  diameter  on  a  steam  bath,  dry  for  two  and  one 
half  hours  in  a  boiling  water  oven,  cool  thoroughly  in  a  desic- 
cator and  then  weigh  quickly. 

Total  Ash 

Char  the  extract  thoroughly  at  a  low  red  heat,  leach  with 
water,  burn  the  insoluble  residue  to  whiteness,  add  the  water 
solution,  evaporate  and  heat  to  low  redness,  cool  in  a  desiccator 
and  weigh. 

Solubility  and  Alkalinity  of  Ash1 

Evaporate  25  cc.  to  dryness,  burn,  cool,  and  weigh ;  extract 
the  ash  repeatedly  on  an  ash-free  filter;  dry  and  ignite  the 
filter  and  residue,  and  weigh  as  insoluble  ash.  Titrate  the 
filtrate,  using  methyl  orange  as  indicator,  and  calculate 
the  number  of  cubic  centimeters  of  tenth-normal  acid  which 
would  be  required  to  neutralize  the  corresponding  filtrate  from 
100  cc.  of  the  vinegar. 

Total  Acidity 

Dilute  10  cc.  in  a  beaker  until  the  solution  appears  very 
light-colored  against  a  white  background,  add  phenolphthalein 
and  titrate  with  standard  sodium  hydroxide.  If  only  the 
total  acidity  is  determined,  the  result  is  expressed  as  acetic  acid. 

Volatile  Acids 

Heat  15  cc.  of  the  vinegar  to  boiling  in  a  flask,  adding  a 
little  tannin  to  check  foaming,  if  necessary;  lower  the  flame 
and  distill  with  steam  until  the  distillate  no  longer  contains 
acid.  Titrate  the  distillate  with  standard  sodium  hydroxide 
and  calculate  as  acetic  acid. 

The  difference  between  the  total  acidity  and  that  due  to 
volatile  acids  gives  a  measure  of  the  fixed  acids  and,  in  the 
case  of  cider  vinegar,  is  calculated  as  malic  acid. 

1  Smith's  method  modified  by  Frear :  J.  Am.  Chem.  Soc.,  1898,  20,  5. 


VINEGAR  AND  ACETATE  129 

Detection  of  Free  Mineral  Acid 

Dilute  5  cc.  of  the  vinegar  with  5  to  10  cc.  water  to  reduce 
the  acidity  to  about  2  per  cent  of  acetic  acid,  add  four  or  five 
drops  of  an  aqueous  solution  of  methyl  violet  (one  part  of 
"methyl  violet  2B"  —  No.  56  of  Bayer  Farbenfabrik,  Elber- 
feld — in  10,000  parts  of  water).  Mineral  acids  change  the 
blue  violet  color  to  a  blue  green  or  green. 

Determination  of  Free  Mineral  Acid  —  Hehner's  Metliod 

To  a  weighed  quantity  of  the  sample,  add  a  measured 
amount  (more  than  sufficient  to  neutralize  all  mineral  acid 
present)  of  tenth-normal  alkali;  evaporate  to  dryness,  incin- 
erate, and  titrate  the  ash  with  tenth-normal  acid,  using  methyl 
orange  as  indicator.  The  difference  between  the  volume  of 
alkali  added  and  that  of  acid  required  represents  the  equiva- 
lent of  the  free  mineral  acid  in  the  sample. 

ACETIC   ACID  AND   ACETATE 

Pure  acetic  acid  is  a  colorless  liquid  of  1.056  specific  gravity, 
miscible  in  all  proportions  with  water,  alcohol,  and  ether. 
When  nearly  anhydrous  it  solidifies  at  about  16°  —  hence  the 
term '"  glacial "  as  applied  to  very  strong  acetic  acid.  The 
pure  acid  boils  without  decomposition  at  119°.  From  dilute 
aqueous  solutions  it  distills  readily  with  steam  at  the  tempera- 
ture of  boiling  water.  The  principal  source  of  commercial 
acetic  acid  is  the  crude  acetate  of  lime  made  by  neutralizing 
the  acid  obtained  in  the  dry  distillation  of  wood.  For  the 
determination  of  the  volatile  acid  obtainable  from  commercial 
acetate  the  following  method  can  be  used. 

DETERMINATION  OF  ACETIC  ACID  IN  CALCIUM  ACETATE 

Arrange  a  round-bottomed  flask  of  300  cc.  capacity  in  such 
a  way  that  it  can  be  inclined  at  an  angle  of  about  60°  from  the 
perpendicular  and  connected  with  a  nearly  vertical  condenser, 
while  another  tube  passing  through  the  stopper  of  the  flask 
provides  for  the  continuous  introduction  of  water,  drop  by 


130  METHODS   OF   ORGANIC    ANALYSIS 

drop,  during  the  distillation.  The  flow  of  water  can  be  con- 
trolled by  means  of  a  screw  pinchcock  or  a  small  dropping 
funnel. 

Weigh  2  grams  of  the  ground  sample,1  transfer  to  the  flask,  and 
add  15  cc.  of  50  per  cent  phosphoric  acid  and  25  cc.  of  water, 
being  sure  that  the  water  washes  down  any  of  the  sample  or  acid 
which  may  have  adhered  to  the  neck  of  the  flask.  Connect  the 
flask  with  the  condenser  and  distill,  collecting  the  distillate  in  a 
receiver  containing  50  cc.  of  water  to  prevent  loss  due  to  evap- 
oration of  acetic  acid.  During  the  distillation  keep  the  volume 
of  liquid  nearly  constant  at  40  cc.  by  admitting  water  free  from 
carbon  dioxide,  in  such  a  way  that  the  drops  fall  upon  the 
inner  surface  of  the  neck  of  the  flask  and  not  directly  into  the 
boiling  liquid.  Continue  the  distillation  until  the  distillate  is 
no  longer  acid.  This  usually  requires  about  one  and  one  half 
hours.  Titrate  the  distillate  with  freshly  standardized  sodium 
hydroxide  solution,  using  phenolphthalein  as  indicator.  Cal- 
culate the  total  acidity  as  percentage  of  acetic  acid  in  the 
sample. 

Notes.  —  It  has  been  found  that  small  amounts  of  phosphoric 
acid  are  frequently  carried  over  mechanically  if  the  acetic  acid 
is  removed  by  a  current  of  steam  or  if  the  distillation  is  con- 
ducted in  an  upright  flask,  especially  when  drops  of  water  are 
allowed  to  fall  directly  into  the  boiling  acid  mixture.  The 
directions  for  arrangement  of  apparatus  are  intended  to  avoid 
this  source  of  error. 

The  phosphoric  acid  used  must  not  contain  nitric  or  any 
other  volatile  acid.  The  large  excess  recommended  dissolves 
the  calcium  phosphate  formed  and  thus  prevents  bumping. 
Oxalic  may  be  substituted  for  phosphoric  acid,  and  the  calcium 
oxalate  removed  by  filtration  before  distilling.  This  method, 
however,  is  longer  and  no  more  accurate  than  the  phosphoric 
acid  method  as  described.  Sulphuric  acid  cannot  be  used,  as  it 
would  be  partially  reduced  to  sulphurous  acid  by  the  tarry 
matter  present  in  the  crude  acetate. 

1  In  sampling,  grinding,  and  weighing  portions  for  analysis,  special  care  must 
be  taken  to  avoid  changes  in  moisture  content. 


VINEGAR  AND   ACETATE  131 

The  distillate  obtained  as  described  should  contain  only  a 
minute  amount  of  carbonic  acid.  It  is  frequently  recommended 
that  the  distillate  be  caught  in  standard  alkali  nearly  sufficient 
to  neutralize  all  the  volatile  acid  expected.  In  this  case  the 
distillate  would  contain  all  carbonic  acid  liberated  in  the  distil- 
lation. The  distillate  containing  some  free  acetic  acid  should 
therefore  be  boiled  under  a  reflux  condenser  to  expel  carbonic 
acid  before  making  the  final  titration.  Errors  may  also  be 
caused  by  variation  in  the  amount  of  carbonate  in  the  alkali 
used  for  titration.  Hence  this  must  be  freshly  standardized, 
using  phenolphthalein  as  indicator,  under  the  same  conditions 
of  temperature  and  removal  of  carbon  dioxide  as  exist  in  the 
titration  of  the  distillate. 

The  volatile  organic  acid,  though  calculated  as  acetic,  always 
contains  some  formic,  propionic,  and  butyric  acids.  In  addition 
to  these  Scheuer  found  small  amounts  of  valerianic,  caproic,  hep- 
tylic,  -caprylic,  and  nonylic  acids.  In  view  of  the  danger  of 
phosphoric  acid  being  carried  over,  it  is  advisable,  after  titrat- 
ing the  distillate,  to  add  nitric  acid,  evaporate  to  25  cc.,  and 
test  for  phosphoric  acid  by  adding  ammonium  nitrate  and  molyb- 
date  solution. 

The  distillation  method  has  now  almost  entirely  replaced  the 
indirect  methods  formerly  used.  There  is,  however,  no  gen- 
eral agreement  as  to  the  details  of  manipulation.  A  full  descrip- 
tion of  the  method  as  used  in  the  laboratory  of  the  General 
Chemical  Company  is  given  by  Grosvenor. 


REFERENCES 

I 

ALLEN  :   Commercial  Organic  Analysis. 

BRANNT  :   Treatise  of  the  Manufacture  of  Vinegar. 

LEACH  :   Food  Inspection  and  Analysis. 

LUNGE  :   Chemisch-technische  Untersuchungsmethoden. 

POST  :   Chemisch-technische  Analyse. 

SUTTON  :   Volumetric  Analysis. 

U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  107. 


132  METHODS  OF  ORGANIC  ANALYSIS 

II 

1899.  CRAMPTON  and  SIMONS  :   Detection  of  Caramel  in  Spirits  and  Vine- 

gar.    J.  Am.  Chem.  Soc.,  21,  16. 

1900.  DOOLITTLE  and  HESS  :    Cider  Vinegar,  its  Solids  and  Ash.     J.  A  m. 

Chem.  Soc.,  22,  218. 

1901.  BROWNE  :   The  Apple  and  Some  of  its  Products.     J.  Am.  Chem.  Soc., 

23,  869. 

1902.  FREAR  :   Vinegar.     U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  65. 
SCHKUER:   Analyse  von  Graukalk  (Calcium  Acetate).     Dissertation. 

Munich. 

1903.  BROWNE  :   Effects  of  Fermentation  upon  the  Composition  of  Cider 

and  Vinegar.     J.  Am.  Chem.  Soc.,  25,  16. 

1904.  GROSVENOR  :   Analysis   of   Commercial    Acetate   of   Lime.     J.   Soc. 

Chem.  Ind.,  23,  530. 
LEACH  and  LYTHGOE  :   Cider  Vinegar  and  Suggested  Standards  of 

Purity.     /.  Am.  Chem.  Soc.,  26,  375. 
STILLWELL  :   Acetic  Acid  (Determination)  in  Acetate  of  Lime.     J. 

Soc.  Chem.  Ind.,  23,  305. 

VAN  SLYKE  :   A  Study  of  the  Chemistry  of  Home-made  Cider  Vine- 
gar.     Bui.    258,    New    York    State    Agricultural    Experiment 

Station. 
1907.   DUBOIS  :   The  Fuller's  Earth  Test  for  Caramel  in  Vinegar.     /.  Am. 

Chem.  Soc.,  29,  75. 
RATCLIFF  :   The   Composition  of   English   Fermentation  Vinegars. 

Analyst,  32,  85. 
WOODMAN  and   SHINGLER:    The   Composition  of   American    Malt 

Vinegar.     Technology  Quarterly,  19,  404. 

1909.   BARKER  and  RUSSEL  :    The  Composition  of  Cider.     Analyst,  34,  125. 
GLADDING  :   The  Analysis  of  Commercial  Acetate  of  Lime.    J.  Ind. 

Eng.  Chem.,  1,  250. 
1910-11.    BALCOM  :   Reports  on  Vinegar.     U.  S.  Dept.  Agriculture,  Bur. 

Chem.,  Bui.  132,  p.  93,  and  Bui.  137,  p.  57. 
1911.   MOTT  :    Cider  Vinegar.     J.  Ind.  Eng.  Chem.,  3,  747. 

Ross :   Determination   of    Glycerol   in    Vinegar    and   Characteristic 

Vinegar  Ratios.     U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  137, 
.  p,  61. 
U<  S.  Dept.  Agriculture,  Office  of  the  Secretary,  Notice  of  Judgment 

No.  1159.     Adulteration  and  Misbranding  of  Vinegar. 


CHAPTER   VII 
Fatty  Acids 

WITH  a  few  possible  exceptions  the  fatty  acids  are  all  mono- 
basic and  those  of  the  common  fats  all  contain  an  even  number 
of  carbon  atoms  in  the  molecule.  Fatty  acids  containing  odd 
numbers  of  carbon  atoms  are  not  widely  distributed  in  nature, 
being  usually  characteristic  of  some  particular  fat  or  small 
group  of  fats.  The  following  statement  of  analytical  proper- 
ties is  based  mainly  upon  the  data  given  by  Lewkowitsch. 

ACIDS  OF  THE  SERIES  CnH2nO2 

Butyric  acid,  C4H8O2,  occurs1  in  butter  and  in  very  small 
quantity  in  a  few  other  fats.  It  is  a  mobile  liquid,  mixing  in 
all  proportions  with  water,  alcohol,  and  ether.  It  boils  with- 
out decomposition  at  162°  and  is  readily  volatile  with  steam. 
Specific  gravity  at  20°,  0.959. 

Caprine  acid,  C6H12O2,  occurs  in  butter  and  coconut  fat.  It 
is  a  rather  oily  liquid  of  unpleasant  odor,  not  miscible  with  water, 
but  somewhat  soluble  in  it,  and  volatile  with  steam.  Boiling 
point  about  200°.  Specific  gravity  at  20°,  0.9241 

Caprylic  acid,  C8H16O2,  occurs  in  coconut  oil,  butter,  and 
human  fat.  In  the  cold  it  crystallizes  in  leaflets  which  melt  at 
16,5°.  It  boils  at  237°  and  is  volatile  with  steam.  One  part 
dissolves,  in  400  parts  of  boiling  water,  from  which  it  separates 
almost  completely  on  cooling.  It  is  readily  soluble  in  alcohol 
and  ether.  Specific  gravity  at  20°,  0.910. 

Oapric  acid,  C10H20O2,  has  been  found  chiefly  in  coconut  oil, 
butter,  and  the  fat  of  the  spice  bush,  Lindera  benzoin.  It  melts 

1  These  statements  refer  to  the  occurrence  of  the  acids  as  esters  rather  than 
in  the  free  state. 

133 


134  METHODS   OF   ORGANIC    ANALYSIS 

at  31.3°,  boils  at  270°,  and  distills  with  steam.  Soluble  in  about 
1000  parts  of  boiling  water ;  almost  insoluble  in  cold  water  ; 
soluble  in  alcohol  and  ether. 

Laurie  acid,  C12H24O2,  occurs  abundantly  in  the  fat  of  the 
seeds  of  Lindera  benzoin,  and  in  smaller  proportions  in  coconut 
fat,  palm  oil,  laurel  oil,  butter,  and  the  nut  oil  of  the  Cali- 
fornia bay  tree.  Laurie  acid  melts  at  43.6°  and  cannot  be  dis- 
tilled at  atmospheric  pressure  without  decomposition.  It  is 
practically  insoluble  in  cold  water,  slightly  soluble  in  boiling 
water,  and  appreciably  volatile  with  steam.  Specific  gravity  at 
20°,  0.883. 

Myristic  acid,  C14H28O2,  is  found  in  nutmeg  butter,  dika  fat, 
butter,  coconut  oil,  lard,  and  many  other  fats,  as  well  as  in 
spermaceti  and  wool  wax.  It  crystallizes  in  leaflets  which  melt  at 
53.8°.  Myristic  acid  is  insoluble  in  water  and  only  very  slightly 
volatile  with  steam.  It  is  not  readily  soluble  in  cold  alcohol. 

Palmitic  acid,  C16H32O2,  occurs  in  nearly  all  solid  fats  and 
non-drying  oils  as  well  as  in  several  waxes  including  spermaceti 
and  beeswax.  It  melts  at  62.6°  and  solidifies  on  cooling  to  a 
scaly  crystalline  mass.  It  is  insoluble  in  water  and  not  readily 
soluble  in  cold  95  per  cent  alcohol,  but  dissolves  in  about  10 
parts  of  cold  absolute  alcohol. 

Stearic  acid,  C18H36O2,  is  found  in  most  fats  and  occurs  most 
abundantly  in  those  having  high  melting  points.  It  crystallizes 
in  leaflets  which  melt  at  69.3°.  It  is  less  soluble  in  alcohol 
than  palmitic  acid,  requiring  about  40  parts  of  absolute  alcohol 
in  the  cold.  It  dissolves  readily  in  ether,  benzol,  carbon  bisul- 
phide, or  hot  alcohol. 

Arachidic  acid,  C20H40O2,  occurs  in  arachis  (peanut)  oil,  and 
has  been  obtained  in  very  small  quantities  from  several  other 
fats.  Arachidic  acid  is  distinguished  from  all  its  lower  homo- 
logues  by  its  insolubility  in  cold  alcohol.  It  dissolves  freely  in 
hot  alcohol,  but  on  cooling  separates  almost  completely  as 
needles  or  crystalline  scales,  which  melt  at  77°  to  78°. 

Behenic  acid,  C22H44O2,  lignoceric  acid,  C24H48O2,  and  an 
isomer  of  the  latter,  carnaubic  acid,  occur  respectively  in  oil  of 
ben,  arachis  oil,  and  carnaiiba  wax. 


FATTY   ACIDS  135 

Cerotic  acid,  C26H52O2,  and  melissic  acid,  C30H60O2,  are  found 
in  the  free  state  in  beeswax. 

The  change  in  properties  of  these  acids,  as  the  molecular 
weight  increases,  is  noticeably  regular.  The  melting  and 
boiling  points  rise  while  the  specific  gravities  and  solubilities 
decrease.  In  general  the  properties  of  the  glycerides  and  other 
esters  vary  in  the  same  way  as  those  of  the  free  acids. 

With  the  exception  of  caproic,  all  of  the  acids  of  this  series, 
occurring  in  natural  fats,  are  believed  to  be  of  the  "  normal " 
(straight  chain)  structure. 

ACIDS  OF  THE  SERIES  CnH2n_2O2 

These  acids  are  unsaturated.  Each  molecule  contains  one 
ethylene  linkage  or  double  bond,  and  is,  therefore,  capable  of 
taking  up  by  direct  addition  two  atoms  of  halogen  or  one  mole- 
cule of  hydrobromic  acid  to  form  a  saturated  compound. 
Careful  oxidation  in  the  presence  of  moisture  results  in  the 
formation  of  the  corresponding  dihydroxy  compounds.  The 
unsaturated  acids  have,  as  a  rule,  lower  melting  points  than  the 
saturated  acids  containing  the  same  number  of  carbon  atoms, 
and  are  therefore  found  more  largely  in  oils  and  soft  fats. 
Only  the  more  important  members  of  the  series  will  be  con- 
sidered here. 

Hypogceic  acid,  ^io^3QO2,  obtained  from  arachis  (peanut)  oil, 
melts  at  33°,  but  is  converted  by  the  action  of  nitrous  acid  into 
the  isomeric  gaidic  acid  which  melts  at  39°.  Phycetoleic  acid, 
isomeric  with  hypogseic,  is  obtained  from  sperm  oil  and  seal  oil. 
It  melts  at  30°  and  is  not  changed  by  nitrous  acid. 

Oleic  acid,  C18H34€2,  occurs  in  nearly  all  fats  and  fatty  oils. 
It  is  an  oily  liquid  which  solidifies  at  4°  and  melts  at  14°. 
Nitrous  acid  converts  oleic  acid  into  the  isomeric  elaidic  acid, 
which  is  a  crystalline  solid  melting  at  44.5°  (Lewkowitsch). 

Erucic  add,  C22H42O2,  found  in  rape  seed  and  mustard  seed 
oils,  melts  at  33°  to  34°.  By  the  action  of  nitrous  or  sulphur- 
ous acid  it  can  be  converted  into  brassidic  acid,  melting  at  65°. 
This  change,  however,  takes  place  less  readily  than  the  corre- 
sponding conversion  of  oleic  acid  into  elaidic  acid. 


136  METHODS   OF   ORGANIC   ANALYSIS 

The  gradual  change  in  properties  with  increasing  molecular 
weight,  noticed  in  the  saturated  acids,  is  not  apparent  in  this 
series,  doubtless  because  the  known  acids  of  the  series  differ  as 
regards  the  position  of  the  double  bond  and  are  therefore  not 
strictly  similar  in  constitution.  The  property  of  changing  to 
solid  isomers  under  the  influence  of  nitrous  acid  is  characteristic 
of  the  acids  of  this  series  and  furnishes  a  means  of  testing  for 
them  in  fatty  oils.  Any  of  the  latter  which  contain  large 
amounts  of  these  acids  become  solid  or  semisolid  on  treatment 
with  nitrous  acid.  This  is  known  as  the  elaidin  test,  and 
is  sometimes  useful  in  the  preliminary  examination  of  oils, 
but  has  been  shown  to  be  less  reliable  than  was  formerly 
supposed. 

An  important  characteristic  of  the  unsaturated  acids  is  the 
solubility  of  the  lead  soaps  in  ether.  Muter's  method  for  the 
separation  of  saturated  and  unsaturated  fatty  acids  is  to  pre- 
cipitate the  mixture  of  acids  as  lead  soaps  and  wash  with  ether. 
While  this  method  is  not  strictly  quantitative,  it  is  probably 
the  best  which  has  been  devised  for  the  purpose. 

ACIDS  OF  THE  SERIES  CnH2n_4O2 

The  acids  of  this  series  unite  with  four  atoms  of  bromine  or 
iodine  and  absorb  oxygen  on  exposure  to  the  air. 

Linoleic  acid,  C18H32O2,  is  the  only  important  member  of  the 
series.  It  is  widely  distributed,  occurring  most  abundantly  in 
the  "  drying  oils,"  so  called  because  they  are  oxidized  to  solids 
on  exposure  to  air.  While  especially  characteristic  of  the 
drying  and  semidrying  oils,  linoleic  acid  is  also  found  in  non- 
drying  oils  and  in  small  quantity  in  some  of  the  solid  fats. 
It  absorbs  oxygen  from  the  air  so  readily  as  to  interfere  se- 
riously with  ordinary  methods  of  purification  and  examination. 
Its  physical  properties  are  therefore  not  well  known,  but  it  has 
been  shown  to  have  a  higher  specific  gravity  than  oleic  acid  and 
a  much  lower  melting  point  since  it  does  not  solidify  at  —  18°. 
Linoleic  acid  is  not  changed  by  nitrous  acid.  Lead  linoleate  is 
soluble  in  ether. 


FATTY   ACIDS  137 

ACIDS  OF  THE  SERIES  CnH2n_6O2 

Linolenic  acid,  C18H30O2,  is  the  only  member  of  this  series 
which  has  been  obtained  in  the  free  state.  It  is  a  colorless  oil 
which  absorbs  oxygen  very  rapidly  from  the  air,  at  the  same 
time  becoming  dark  brown.  The  purest  preparations  of  the 
acid  which  have  been  described  absorbed  242  to  245  per  cent 
of  iodine.  The  pure  acid  should  absorb  six  atoms  or  274  per 
cent  of  iodine.  Linolenic  acid  has  not  been  solidified.  Its 
specific  gravity  is  greater  than  that  of  linoleic  acid.  The  lead 
soap  is  soluble  in  ether.  Linolenic  acid  occurs  in  considerable 
quantity  in  linseed  oil.  By  oxidizing  linseed  oil  acids  with 
alkaline  permanganate,  Hazura  obtained  two  acids  of  the  formula 
C18H30(OH)6O2,  linusic  and  isoliriusic  acids,  and  hence  inferred 
the  presence  of  an  isolinolenic  acid  in  linseed  oil.  Jecoric  acid, 
isomeric  with  linolenic  acid,  is  reported  by  Fahrion 1  as  existing 
in  sardine  oil.  The  highly  unsaturated  acids  of  fish  and  marine 
animal  oils  are  in  general  isomeric,  rather  than  identical,  with 
those  of  linseed  oil. 

ACIDS  OF  THE  SERIES  CnH2n_8O2 

Five  acids  have  been  reported  as  belonging  to  this  series. 
The  only  one  which  need  be  mentioned  here  is  clupanodonic 
acid,  C18H28O2,  occurring  in  Japanese  sardine  oil,  herring  oil, 
and  whale  and  turtle  oils. 

SATURATED  HYDROXY-ACIDS 

.  When  any  of  the  unsaturated  acids  occurring  in  vegetable 
oils  is  carefully  oxidized  in  alkaline  solution  by  means  of 
potassium  permanganate,  hydroxyl  is  added  until  the  mole- 
cule becomes  saturated.  Thus  oleic  acid  yields  dihydroxy- 
stearic  acid,  C18H34(OH)2O2,  linoleic  acid  yields  sativic 
acid,  C18H32(OH)4O2,  and  linolenic  acid  yields  linusic  acid, 
C18H30(OH)6O2.  Similar  changes  in  the  unsaturated  acids 
may  take  place  when  their  glycerides  are  exposed  to  the  air. 
Small  amounts  of  saturated  hydroxy-acids  are,  therefore,  likely 
1  Chem.  Ztg.,  1893,  17,  521. 


138  METHODS   OF   ORGANIC   ANALYSIS 

to  be  present  in  fatty  oils  which  have  been  kept  for  some  time 
in  partially  filled  bottles  or  otherwise  exposed  to  atmospheric 
oxidation.  Only  two  acids  of  this  series  have  as  yet  been 
found  in  nature.  Juillard1  found  about  1  per  cent  of  dihy- 
droxystearic  acid  in  castor  oil  and  Browne  2  the  same  quantity 
in  butter  fat.  A  dihydroxy-acid  of  the  formula,  C30H60O4,  is 
said  to  occur  in  wool  wax  3  and  is  called  lanocerinic  or  lanoceric 
acid. 

HYDROXY-ACID  OP  THE  SERIES  CnH2n_2O3 

Ricinoleic  acid,  C18H34O3,  the  only  important  member  of  this 
series,  occurs  in  large  quantity  in  castor  oil.  It  is  a  very 
viscous  liquid  of  much  higher  specific  gravity  than  oleic  or 
linoleic  acid.  According  to  Juillard4  the  pure  acid  melts  at 
4°-5°,  and  is  miscible  with  alcohol  and  ether  in  all  proportions. 
Ricinoleic  acid  is  dextrorotatory.  The  position  of  the  asym- 
metric carbon  atom,  as  well  as  of  the  double  bond,  is  shown 
by  the  following  formula  :  5 

C6H13  •  CH(OH)  •  CH2  •  CH  :  CH  .  (CH2)7  •  COOH. 

Ricinoleic  resembles  oleic  acid  in  its  chemical  reactions.  It 
absorbs  two  atoms  of  bromine  or  iodine.  By  treatment  with 
permanganate  in  alkaline  solution  two  hydroxyl  radicles  are 
added.  By  treatment  with  nitrous  acid,  ricinoleic  acid  is  con- 
verted into  the  solid  isomer,  ricinelaidic  acid.  Lead  ricin- 
oleate  is  easily  soluble  in  ether. 

SEPARATION  OF  FATTY  ACIDS 

For  the  analytical  separation  of  saturated  from  unsaturated 
fatty  acids,  Muter's  method,  based  on  the  solubility  of  the  lead 
soaps  of  the  latter  in  ether,  is  generally  used.  In  order  to 
determine  the  constituents  of  a  mixture  of  homologous  acids 

1  Bull.  Soc.  Chim.,  1895,  [3],  13,  238. 

2  J.  Am.  Chem.  Soc.,  1899,  21,  817. 

8  Darmstaedte?^and  Lifschiitz:  Ber.,  1896,  29,  1476. 
4  Bull.  Soc.  Chim.,  1895,  [3],  13,  240. 

6  Goldsobel :  Ber.,  1894,  27,  3121.  Kasansky :  J.  prakt.  Chem.,  1900,  62, 
363. 


FATTY   ACIDS  139 

it  is  usually  necessary  to  fraction  the  mixture  repeatedly  either 
by  distillation,  precipitation,  or  crystallization  until  each  frac- 
tion contains  only  two  acids.  The  proportions  of  the  two  con- 
stituents can  then  be  found  by  determining  the  mean  molecular 
weight  of  the  mixture.  This  is  usually  done  in  a  manner  simi- 
lar to  the  determination  of  the  saponification  number  or  saponi- 
fication  equivalent  as  described  in  the  next  chapter. 

REFERENCES 
I 

ALLEN  :   Commercial  Organic  Analysis. 

BENEDIKT  and  ULZER  :   Analyse  der  Fette  und  Wachsarten. 

LEATHES  :    The  Fats. 

LEWKOWITSCH:   Chemical  Technology  and  Analysis  of  the  Oils,  Fats,  and 

Waxes. 

OPPENHEIMER  :    Handbuch  der  Biochemie. 
ULZER  and  KLIMONT  :   Allgemeine  und  Physiologische  Chemie  der  Fette. 

II 

1899.   BROWNE:    (Separation  of  the  Acids  of  Butter  Fat).     J.  Am.  Chem. 

Soc.,  21,  807. 
1906.    HALLER:    (Separation  of  Fatty  Acids).     Compt.  rend.,  143,  657. 

1908.  DUNHAM  :    Carnaiibic  Acid  from  Beef  Kidneys.     Proc.  Soc.  Exper. 

Biol  Med.,  5,  58. 

1909.  ERDMANN,  BEDFORD  and   RASPE  :   Constitution  of  Linolenic  Acid. 

Ber.,  42,  1334. 
HARTLEY:    Nature  of  the  Fat  contained  in  the  Liver,  Kidney,  and 

Heart.     /.  PhysioL,  38,  353. 
RALLETT  :    (On  Linoleic  and  Linolenic  Acids).     Z.  physiol.  Chem., 

62,  410,  422. 
TOLMAN  :   A  Study  of  the  Fatty  Acids  of  Fish  Oils.     /.  Ind.  Eng. 

Chem.,  1,  340. 

1910.  ROSAUER  :    The  Manufacture  and  Examination  of   Technical  Oleic 

Acid.     Chem.    Rev.  Fette-Harz-Ind.,  18,  28, 43 ;    Chem.  Abs.,    5, 
1683,  1846. 

1911.  HOLLAND  :    Purification  of    Insoluble   Fatty   Acids.     J.   Ind.   Eng. 

Chem.,  3,  171. 


CHAPTER   VIII 
Oils,  Fats,  and  Waxes  —  General  Methods 

ALL  the  glycerides  of  fatty  acids  are  known  as  fats.  As  a 
matter  of  convenience  those  fats  which  are  liquid  at  ordinary 
temperatures  are  commonly  called  fatty  oils.  Fats  and  fatty 
oils,  therefore,  consist  of  a  definite  group  of  compounds,  the 
glyceryl  esters  of  the  fatty  acids.  In  any  given  natural  fat  a 
number  of  glycerides  may  be  found,  as  is  evident  from  the 
statements  regarding  sources  of  the  various  fatty  acids  in  the 
last  chapter. 

Waxes  are  esters  of  fatty  acids  with  monatomic  alcohols  of 
high  molecular  weight.  Most  waxes  are  solid  at  ordinary 
temperatures  and  the  term  is  sometimes  applied  to  other  solids 
of  similar  physical  properties,  solid  hydrocarbons,  for  example, 
being  frequently  called  mineral  waxes.  Among  the  esters 
found  in  some  of  the  common  waxes  are :  cetyl  palmitate  in 
spermaceti ;  myricyl  palmitate  in  beeswax ;  dodecatyl  oleate 
in  sperm  oil. 

The  term  "  oil "  has  no  strict  chemical  significance,  being 
applied  not  only  to  liquid  fats,  but  also  to  substances,  such  as 
mineral  and  essential  oils,  which  are  similar  in  some  physical 
properties  but  entirely  different  in  constitution.  The  mineral 
oils  are  conveniently  treated  in  connection  with  the  fatty 
oils  in  works  on  technical  analysis,  because  on  account 
of  their  similar  physical  properties  they  may  for  certain 
uses  be  mixed  with,  or  substituted  for,  J;he  fatty  oils  and 
are  sometimes  found  as  adulterants  of  the  latter.  The  same 
is  true  of  some  of  the  essential  oils,  notably  rosin  oil  and 
turpentine. 

The  methods  given  in  this  chapter  are  described  and  dis- 

140 


OILS,    FATS,    AND    WAXES  141 

cussed  with  special  reference  to  the  analysis  of  solid  and  liquid 
fats.  In  most  cases,  however,  they  are  also  applicable  to  waxes, 
non-fatty  oils,  and  technical  mixtures. 

CLASSIFICATION 

Lewkowitsch  classifies  chiefly  according  to  capacity  for  ab- 
sorbing iodine.  "  Drying  "  oils  show  high  iodine  absorption  ; 
"  non-drying  "  oils,  low  ;  "  semidrying  "  oils,  intermediate. 

In  Allen's  Commercial  Organic  Analysis  the  solid  and  liquid 
fats  and  waxes  are  classified  in  twelve  groups  essentially  as 
follows : 

I.  Olive  oil  group  —  vegetable  fatty  oils  consisting  chiefly  of 
olein,  with  smaller  amounts  of  palmitin,  stearin,  and  perhaps  other 
saturated  glycerides,  and  containing  also,  at  least  in  some  cases, 
linolein  or  similarly  unsaturated  glycerides  in  small  amount. 

II.  Rape  oil  group  —  fatty  oils  from  the  seeds  of  the  crucif- 
erce,  characterized  by  containing  erucin. 

III.  Cottonseed  oil  group  (semidrying  oils)  —  consisting  largely 
of  olein  arid  linolein  with   small   quantities   of   palmitin   and 
stearin  and  traces  of  linolenin. 

IV.  Linseed  oil  group  (drying  oils) — differing  in  composition 
from  group  III  in  containing  larger  proportions  of  linolein  and 
linolenin. 

V.  Castor  oil  group  —  in  some  at  least  of  which  (e.g.  castor 
and  grapeseed  oils)  ricinolein  predominates. 

VI.  Cfacao  butter  group  —  vegetable  fats    consisting  mainly 
of   olein,  stearin,  palmitin,  and  myristin,  with  perhaps    small 
amounts  of  linolein  and  of  the  glycerides  of  saturated  acids  of 
lower  molecular  weight  than  myristic. 

VII.  Coconut  oil  group  —  vegetable  fats  containing  less  olein 
than  those  of  group  VI  and  more   of   the   glycerides   of   the 
saturated  fatty  acids  of  low  molecular  weight,  laurin,  etc. 

VIII.  Lard  oil  group  —  animal  fatty  oils  consisting  mainly 
of   olein  with  smaller  amounts   of   stearin   and   palmitin   and 
probably  some  linolein  (or  isomeric  glyceride). 

IX.  Tallow  group  —  animal  fats  consisting  in  most  cases  chiefly 
of  olein,  stearin,  and  palmitin  with  perhaps  a  little  of  linolein 


142  METHODS   OF   ORGANIC   ANALYSIS 

(or  an  isomer).  Butter  differs  from  the  other  members  of  the 
group  in  containing  butyrin,  laurin,  and  myristin  with  smaller 
amounts  of  caproin,  caprylin,  and  caprin,  and  very  little  stearin. 

X.  Whale  (and  fish)  oil  group  —  fatty  oils  of  marine  animals, 
including  fish,  consisting  largely  of  glycerides  of  acids  isomeric 
with  linoleic  and  linolenic. 

XI.  Sperm  oil  group  —  liquid  waxes. 

XII.  /Spermaceti  group  —  solid  waxes   of   both   animal   and 
vegetable  origin. 

PROPERTIES  OF   FATS   AND  FATTY  OILS 

Refined  fats  and  fatty  oils  are  usually  light  yellow  to  color- 
less. Vegetable  oils  are  sometimes  tinged  green  by  the  pres- 
ence of  chlorophyll.  Crude  oils  are  often  reddish  or  even  dark 
brown.  The  characteristic  colors,  odors,  and  flavors  of  natural 
fats  are  due  to  small  quantities  of  substances  other  than 
glycerides,  and  therefore  become  less  perceptible  the  more 
thoroughly  the  oil  is  refined.  It  is  believed  that  all  of  the 
natural  glycerides  except  butyrin,  if  obtained  absolutely  pure, 
would  be  colorless,  tasteless,  and  odorless. 

The  natural  glycerides  are  all  lighter  than  water  and  insoluble 
in  it.  They  can  take  up  a  very  small  amount  of  water,  which 
is  given  off  as  steam  on  heating.  The  quantity  of  water  which 
can  be  held  by  a  fatty  oil  without  causing  turbidity  is,  however, 
negligible,  so  that  for  practical  purposes  the  fatty  oils  may  be 
considered  as  immiscible  with  water. 

They  dissolve  readily  in  ether,  carbon  bisulphide,  chloroform, 
carbon  tetrachloride,  and  benzol,  and  mix  with  each  other  in  all 
proportions.  With  the  exception  of  castor  oil  and  a  few  other 
oils  characterized  by  a  large  proportion  of  hydroxy-acids,  they 
are  sparingly  soluble  in  alcohol  or  acetic  acid,  but  dissolve 
readily  in  petroleum  ether  and  mix  in  all  proportions  with 
mineral  oils.  Castor  oil  is  readily  soluble  in  alcohol  or  acetic 
acid  and  not  readily  miscible  with  petroleum  ether  or  mineral  oils. 

The  natural  fats  do  not  distill  without  decomposition.  When 
decomposed  by  heating  they  give  off  acrolein,  which  is  readily 
recognized  by  its  characteristic  irritating  odor. 


OILS,    FATS,    AND   WAXES  143 

A  simple  triglyceride  is  one  in  which  the  three  acid  radicals 
are  of  the  same  kind.  A  glyceryl  ester  containing  the  radicals 
of  two  or  three  different  fatty  acids  is  known  as  a  mixed  glyc- 
eride.  Both  simple  and  mixed  glycerides  have  been  isolated 
from  natural  fats,  and  certain  physical  differences  in  fats  which 
contain  practically  the  same  acids  are  now  attributed  to  the 
presence  of  mixed  glycerides.  For  discussions  of  simple  and 
mixed  triglycerides,  see  Lewkowitsch's  Oils,  Fats,  and  Waxes, 
Chapter  I,  and  recent  papers  by  Hansen,1  Holde,2  and  Kreis 
and  Hafner. 3 

ANALYTICAL  METHODS 

The  object  of  an  ordinary  fat  or  oil  analysis  is  not  so  much 
to  separate  individual  constituents  as  to  determine  certain 
chemical  and  physical  properties  which  are  fairly  constant  for 
each  variety  when  pure,  and  are  therefore  frequently  called 
analytical  constants.  All  solid  and  liquid  fats  being  essentially 
mixtures  of  triglycerides,  any  differences  in  chemical  and 
physical  properties  (except  such  physical  variations  as  are  due 
to  mixed  glycerides)  must  be  attributed  mainly  to  the  presence 
of  different  fatty  acids,  or  of  the  same  acids  in  different 
proportions. 

The  principal  differences  to  be  expected  are :  (1)  In  the 
mean  molecular  weight  of  the  acids  present  or  the  relative  pro- 
portions of  acids  of  high  and  those  of  low  molecular  weight ; 
(2)  in  the  relative  number  of  "double  bonds"  depending  upon 
the  proportions  of  acids  of  the  stearic,  oleic,  linoleic,  and  lino- 
lenic  types  •  (3)  in  the  proportion  of  hydroxy-acids  present. 

Some  of  the  analytical  "constants"  express  direct  measures 
of  one  of  these  three  properties.  Others,  especially  the  physi- 
cal constants,  are  influenced  by  variation  in  any  of  these  three 
directions  and  therefore  express  no  one  chemical  property,  but 
a  resultant  of  all.  The  "  constants  "  most  used  may  be  grouped 
on  this  principle  as  follows : 

1  Ueber  das  Vorkommengemischter  Fettesaiire-Glyceride  in  theirischen  Fette. 
Dissertation,  Rostock,  1902 ;  Arch.  Hygiene,  1902,  42,  1. 
2J5er.,  1902,  35,  4306. 
*Ber.,  1903,  36,  1123,  2766;  Z.  Nahr.  Genussm.,  1904,  7.  641. 


144  METHODS   OF   ORGANIC    ANALYSIS 

1.  (#)  Measuring  the  mean  molecular  weight. — Saponifica- 
tion  or  Koettstorfer  number.      (5)  Measuring  the  proportion  of 
acids  of  high  or  of  low  molecular  weight.  —  Hehner  number, 
Reichert-Meissl  number. 

2.  (a)  Measuring  the  proportion  of  un saturated  acids  (num- 
ber of   "double  bonds").  —  Hubl  number  and  other  halogen 
absorption  numbers.      (£)  Depending  mainly  upon  the  propor- 
tion of  unsaturated  acids.  —  Thermal  reactions  with  bromine  or 
sulphuric  acid,  Maumene  number. 

3.  Measuring  the  hydroxyl  radical  and  therefore,  depend- 
ing   mainly    upon    the    presence    of    hydroxy-acids.  —  Acetyl 
number. 

4.  Influenced   by    all    of    the    above    properties.  —  Specific 
gravity,  index  of  refraction,  melting  point,  "liter  test,"  vis- 
cosity, solubilities. 

THE  SAPONIFICATION   OR   KOETTSTORFER  NUMBER  x 

The  saponification  or  Koettstorfer  number  is  the  number  of 
milligrams  of  potassium  hydroxide  consumed  in  the  complete 
saponification  of  one  gram  of  the  fat  or  wax ;  or,  in  other  words, 
it  is  ten  times  the  percentage  of  potassium  hydroxide  required 
to  neutralize  the  total  fatty  acids  in  the  sample,  whether  free 
or  in  the  form  of  esters. 

Reagents. — 1.  Standard  solution  of  hydrochloric  acid  pref- 
erably half  normal. 

2.  Alcoholic  potash  solution  containing  40  grams  of  potas- 
sium hydroxide  per  liter  of  purified  95  per  cent  alcohol.     In 
the  preparation  of  this  solution  the  best  available  potassium 
hydroxide  (purified  by  alcohol)  should  be  dissolved  in  alcohol 
which  has  been  purified  by  redistillation  over  caustic  alkali. 
The  solution  must  be  clear  when  used  (filter  if  necessary). 

3.  As  indicator  a  1  per  cent  solution  of  phenolphthalein  in 
purified  95  per  cent  alcohol. 

Determination.  —  Weigh  4  to  5  grams  of  the  fat  or  oil  in  a  250- 
cc.  Erlenmeyer  flask,  add  50  cc.  of  the  alcoholic  potash  solution, 

1  Koettstorfer:  Z.  anal,  cheni.,  1879,  18,  199,  431.  Reprinted  in  Ephraim's 
Originalarbeiten  liber  Analyse  der  Nahrungsmittel,  Leipzig,  1895. 


OILS,    FATS,    AND   WAXES  145 

connect  with  a  reflux  condenser  and  boil  for  thirty  minutes,  or 
until  the  oil  is  completely  saponified,  so  that  the  liquid  in  the 
flask  appears  homogeneous  and  clear.  At  the  same  time  meas- 
ure 50  cc.  of  the  alcoholic  potash  solution  into  an  empty  flask 
of  the  same  size  and  shape,  connect  with  a  similar  reflux  con- 
denser and  boil  for  the  same  length  of  time,  and  treat  in  all 
respects  in  exactly  the  same  way  as  in  the  case  of  the  solution 
containing  the  sample.  Cool  the  flasks  and  titrate  each  with 
the  standard  hydrochloric  acid,  using  1  cc.  of  the  phenolphtha- 
lein  solution  as  indicator.  The  difference  between  the  titra- 
tions  gives  a  measure  of  the  potassium  hydroxide  consumed  in 
saponifying  the  sample. 

Notes.  —  When  ordinary  alcohol  is  used  for  the  potassium  hy- 
droxide solution,  the  latter  rapidly  turns  brownish  so  that  the 
final  titration  is  difficult.  Alcohol  which  has  been  treated  with 
potassium  hydroxide,  allowed  to  stand  for  one  to  two  weeks, 
and  then  redistilled  gives  a  much  more  permanent  solution. 
According  to  Gill,  scarcely  any  darkening  of  the  solution  occurs 
if  it  is  kept  under  an  atmosphere  of  hydrogen.  Great  care 
must  be  exercised  to  measure  exactly  the  same  quantity  of  the 
alkaline  solution  into  each  flask,  and  to  treat  the  blank  solution 
in  exactly  the  same  way  as  that  containing  the  sample,  so  that 
any  loss  of  alkalinity  due  to  absorption  of  carbon  dioxide  from 
the  air,  or  to  the  possible  action  of  the  alkali  on  the  solvent, 
may  be  the  same  in  each  case.  If  sulphuric  acid  were  used  in 
place  of  hydrochloric  for  the  final  titration,  a  precipitate  of 
potassium  sulphate  would  be  formed  in  the  alcoholic  solution, 
thus  impairing  the  delicacy  of  the  end  reaction. 

In  order  to  avoid  the  changes  which  may  occur  in  the  alkali 
solution  on  boiling,  Henriques l  recommends  that  the  saponifi- 
cation  be  conducted  in  the  cold.  From  3  to  4  grams  of  oil  are 
mixed  with  25  cc.  of  petroleum  ether  and  25  cc.  of  normal  alco- 
holic potash  and  allowed  to  stand  overnight  at  room  tempera- 
ture, when  the  saponification  is  said  to  be  complete.  A  blank 
test  with  the  same  amounts  of  petroleum  ether  and  alcoholic 
potash  should  be  made  alongside. 

1  Z.  angew.  Chem.,  1895,  721 ;    1896,  221,  423. 


146 


METHODS  OF  ORGANIC  ANALYSIS 


The  Saponification  Equivalent 

The  results  obtained,  as  described  above,  are  sometimes  ex- 
pressed in  terms  of  the  saponification  equivalent.  This  is  the 
weight  of  fat  which  reacts  with  the  molecular  weight  of  sodium 
or  potassium  hydroxide ;  or,  in  other  words,  the  number  of 
grams  of  fat  which  would  be  saponified  by  one  liter  of  normal 
alkali.  Comparing  this  with  the  definition  of  the  saponification 
(or  Koettstorfer)  number,  it  will  be  seen  that  these  two  values 
express  the  same  property  in  reciprocal  terms  and  that  the 
product  of  the  two  values  is  always  equal  to  the  number  of 
milligrams  of  potassium  hydroxide  in  a  liter  of  normal  solution, 
viz.  56,108. 

Hence 

56108 


Saponification  equivalent  = 
Saponification  number  = 


saponification  number 
56108 


sapouification  equivalent 
For  a  sample  consisting  entirely  of  triglycerides  the  saponi- 
fication equivalent  would  be  exactly  one  third  of  the  mean 
molecular  weight  of  these  glycerides,  and  the  mean  molecular 
weight  of  the  fatty  acids  would  be  (C8H2  -s-  3)  or  12.67  units 
lower  than  the  saponification  equivalent. 

TABLE  13.  —  SAPONIFICATION  DATA  OF  SOME  PURE  ESTERS 
(Data  are  given  to  nearest  0.1  only) 


Ester 

Saponification 
number 

Saponification 
equivalent 

Butyrin 

557  7 

100  7 

Lcturin 

263  8 

212  9 

208.8 

268.9 

189.1 

297.0 

Olein     .     

190.4 

294.9 

160.0 

351.0 

191.7 

292.9 

193.0 

290.9 

180.6 

310.9 

Dodecatyl  oleate  1    

124.5 

450.5 

1  Constituent  of  sperm  oil. 


OILS,    FATS,    AND   WAXES  147 

The  natural  fats,  being  mixtures  of  glycerides,  rarely  show 
such  distinct  differences  in  their  saponification  numbers  and 
equivalents  as  the  above  data  might  suggest.  The  saponifi- 
cation number  is,  however,  a  useful  aid  in  detection  of  glycer- 
ides of  acids  below  C16  or  above  C18,  of  unsaponifiable  oils, 
or  of  waxes. 

ACID  AND  ESTER  NUMBERS 

The  acid  number  of  a  fat  or  wax  is  the  number  of  milligrams 
of  potassium  hydroxide  required  to  neutralize  the  free  fatty 
acids  in  one  gram  of  substance.  The  ester  number  is  the  dif- 
ference between  the  saponification  number  and  the  acid  number 
and  therefore  shows  the  amount  of  alkali  consumed  in  the 
saponification  of  esters. 

To  determine  the  acid  number,  shake  4  to  5  grams  of  the 
sample  with  50  cc.  of  carefully  neutralized  warm  alcohol  and 
titrate  with  tenth-normal  or  half-normal  alkali,  using  phenol- 
phthalein  as  indicator.  As  the  oil  itself  mixes  but  slightly 
with  alcohol,  it  is  necessary  toward  the  end  of  the  titration  to 
shake  thoroughly  after  each  addition  of  alkali  to  secure  com- 
plete extraction  of  the  fatty  acid  from  the  oily  layer.  The 
alcohol  should  be  neutralized  immediately  before  using. 

Note.  —  The  acidity  of  a  sample  of  oil  or  fat  is  not  always 
expressed  as  the  "  acid  number."  Frequently  it  is  recorded  in 
terms  of  the  equivalent  percentage  of  free  oleic  acid,  "  acidity 
as  oleic,"  and  sometimes  as  "  degrees  of  acidity,"  which  indi- 
cates the  number  of  cubic  centimeters  of  normal  caustic  alkali 
required  to  neutralize  the  free  acids  in  100  grams  of  the  fat. 
This  latter  form  of  expressing  acidity  is  not  common  and  should 
not  be  encouraged.  The  expressing  of  acidity  sometimes  as 
acid  number  and  sometimes  as  percentage  of  oleic  acid  is  not 
seriously  inconvenient  if  it  be  kept  in  mind  that  in  any  given 
case  the  acid  number  is  almost  exactly  twice  the  percentage  of 
free  acid  calculated  as  oleic. 

For  titrating  dark  fats  the  use  of  Alkali  Blue  6B  as  indicator 
in  place  of  phenolphthalein  has  been  recommended.  About 
2  cc.  of  a  2  per  cent  alcoholic  solution  are  used. 


148  METHODS  OF  ORGANIC  ANALYSIS 

THE  HEHNER  NUMBER 

The  Hehner  number  is  the  percentage  of  insoluble  fatty  acids 
obtainable  from  a  fat. 

As  the  determination  is  ordinarily  made,  the  unsaponifiable 
matter  present  in  the  fat  is  weighed  with  the  insoluble  acids. 
The  great  majority  of  fats  and  fatty  oils  have  Hehner  numbers 
between  94.5  and  96.  Butter  fat  has  a  lower  Hehner  number 
and  the  determination  of  this  "  constant "  is  of  value  chiefly  in 
testing  the  purity  of  butter.  The  detailed  description  of  the 
process  will  therefore  be  given  in  the  section  on  butter  analysis. 

THE  REICHERT-MEISSL  NUMBER 

The  Reichert-Meissl  number  is  the  number  of  cubic  centi- 
meters of  tenth-normal  caustic  alkali  required  to  neutralize  the 
soluble  volatile  acids  obtained  from  5  grams  of  a  fat  by  the 
Reichert  distillation  process. 

This  number  serves  as  a  comparative  measure  of  the  acids  of 
low  molecular  weight.  Its  principal  use  is  in  the  examination 
of  butter  fat  and  the  detailed  description  will  be  given  in  that 
connection.  The  Reichert  number  is  about  one  half  the  Reich- 
ert-Meissl number,  Reichert  having  originally  recommended 
the  use  of  2.5  grams  of  fat.  Among  the  common  oils  and  fats 
the  Reichert-Meissl  numbers  are  usualLy  less  than  1.0.  Butter 
fat  has  a  high  Reichert-Meissl  number  and  as  this  determination 
is  used  principally  in  the  examination  of  butter,  it  will  be  de- 
scribed in  the  section  on  butter  analysis. 

THE  IODINE  OR  HUBL  NUMBER 

The  iodine  or  Hiibl  number  is  the  percentage  of  iodine  (or 
of  iodine  chloride  or  bromide  expressed  in  terms  of  iodine)  ab- 
sorbed by  the  sample. 

This  number  gives  a  quantitative  measure  of  the  unsaturated 
fatty  acids  (or  of  the  "  number  of  double  bonds  ")  in  a  fat  or 
wax. 

Mills,  Snodgrass,  and  Akitt  were  probably  the  first  to  make 


OILS,    FATS,    AND   WAXES  149 

systematic  use  of  the  halogen  absorbing  power  in  fat  analysis. 
In  their  experiments l  the  oil  or  fat  to  be  tested  was  dissolved 
in  carbon  tetrachloride  and  titrated  with  a  standard  solution  of 
bromine  in  carbon  tetrachloride  as  long  as  the  bromine  was  ab- 
sorbed. At  about  the  same  time,  Hiibl  published2  a  method 
based  upon  the  use  of  iodine  in  an  alcoholic  solution  of  mercu- 
ric chloride  which  was  so  carefully  worked  out  in  all  of  its  de- 
tails that  the  original  form  of  the  process  is  still  used  in  many 
laboratories  in  preference  to  any  of  the  modifications  which 
have  been  proposed.  Recently,  however,  the  Wijs 3  and  the 
Hanus  4  modifications  have  been  largely  used,  and  it  is  probable 
that  they  will  gradually  replace  the  Hiibl  process.  These  three 
methods  will  be  given  here.  The  bromine  absorption  method 
as  developed  by  Mcllhiney  5  can  be  used  in  place  of  the  iodine 
methods,  but  is  more  especially  adapted  to  the  examination  of 
linseed  oil  for  rosin  oil  or  rosin,  and  will  be  referred  to  in  that 
connection. 

Method  of  Hull 

Reagents.  —  1.  Iodine  solution.  Dissolve  26  grams  of  pure 
iodine  in  500  cc.  of  95  per  cent  alcohol.  Dissolve  30  grams  of 
mercuric  chloride  in  500  cc.  of  alcohol  of  the  same  strength. 
Mix  the  two  solutions  at  least  12  hours  before  using.  As  this 
solution  is  expensive  and  does  not  keep  well,  no  more  than 
three  days'  supply  should  be  made  at  one  time. 

2.  Standard  solution  of  sodium  thiosulphate.  Dissolve  24 
grams  of  the  crystallized  salt  in  a  liter  of  water,  allow  to  stand 
at  least  24  hours,  and  then  determine  the  strength  of  the  solu- 
tion in  terms  of  iodine.  While  any  of  the  well-known  methods 
may  be  used,  it  is  convenient  to  standardize  the  thiosulphate 
solution  as  follows : 

Weigh  3. 8694  grams  of  pure  dry  potassium  dichromate,  dis- 
solve in  water  and  dilute  to  1000  cc.  In  a  well-filled  tightly 
stoppered  bottle,  this  standard  solution  of  dichromate  can  be 

1  J.  Soc.  Chem.  Ind.,  1883,  2,  435;  1884,  3,  366. 

2  Dingl.  polyt.  J".,  1884,  253,  281.     Reprinted  by  Ephraim,  loc.  cit. 
sBer.,  1898,  31,  750.  *  Z.  Nahr.-Gemtssm.,  1901,  4,  913. 
5  J.  Am.  Chem.  Soc.,  1884,  16,  245;  1899,  21,  1084;  1902,  24,  1109. 


150  METHODS    OF   ORGANIC    ANALYSIS 

kept  indefinitely  without  deterioration.  Each  cubic  centimeter 
of  this  solution  is  equivalent  to  0.01  gram  iodine.  Mix  25  cc.  of 
a  15  per  cent  potassium  iodide  solution  with  5  cc.  of  hydro- 
chloric acid,  add  50  cc.  of  the  dichromate  solution,  and  titrate  the 
liberated  iodine  by  means  of  the  thiosulphate  solution,  observing 
procedure  and  precautions  described  below.  Calculate  the 
amount  of  iodine  consumed  by  each  cubic  centimeter  of  the  thio- 
sulphate solution.  As  a  precaution  the  strength  of  either  the  di- 
chromate or  the  thiosulphate  solution  should  also  be  determined 
by  an  independent  method. 

3.  An  approximately  15  per  cent  solution  of  pure  potassium 
iodide  in  cold,  recently  boiled,  distilled  water. 

4.  Freshly  prepared  starch  solution,  1  part  starch  to  200 
parts  of  water,  for  use  as  indicator. 

5.  Pure  chloroform. 

6.  Cold,  recently  boiled,  distilled  water. 
Determination. — Thoroughly  clean  and  dry  two  or  more  thin 

Erlenmeyer  flasks,  of  the  form  made  for  this  purpose,  having 
accurately  ground  glass  stoppers  and  flaring 
mouths   which    form    a    gutter    between    the 
stopper  and   the   lip,  as   shown   in    Fig.    10. 
Into  one  of  these  flasks  weigh  accurately  about 
0.25  gram  of  the  sample,  and  add  10  cc.  of 
chloroform.     When  the  sample  has  completely 
dissolved,  add  50  cc.  of  the  mixed  iodine  solu- 
tion, stopper  carefully,  fill  the  gutter  around 
the  stopper  with  potassium  iodide  solution  to 
guard  against  loss  of  iodine,  shake  gently,  and 
allow  the  flask  to  stand  in  a  cool  dark  closet 
for  three  hours.     In  another  clean  dry  flask  of 
FIG.  10.— Flask  for  the  same  size  and  form  make  a  blank  deter- 
fwlrnenumber.     '*  mination,  usvng  the  same  amounts  of  chloro- 
form, iodine  solution,  and   potassium  iodide. 
Allow  the  two  flasks  to  stand  side  by  side  for  the  same  length 
of  time.     When  the  absorption  is  complete,  lift  the  stopper  in 
such  a  way  that  its  lower  surface  will  be  washed  by  the  iodide 
solution  from  the  gutter;  add  100  cc.  of  cold,  recently  boiled, 


OILS,    FATS,    AND   WAXES  151 

distilled  water  and  20  cc.  more  of  the  potassium  iodide  solution, 
washing  down  the  sides  of  the  flask  with  the  latter.  In  case  a 
red  precipitate  of  mercuric  iodide  appears,  add  more  potassium 
iodide  until  the  precipitate  is  dissolved.  Titrate  the  excess  of 
iodine  at  once  by  means  of  the  standard  thiosulphate  solution. 
The  latter  may  be  run  in  rapidly  until  the  iodine  is  nearly  con- 
sumed and  the  solution  is  only  light  yellow;  then  add  2  cc.  of 
the  starch  solution  and  finish  the  titration  carefully  but  with- 
out delay.  As  the  end  point  is  approached,  stopper  the  flask 
quickly  after  each  addition  of  thiosulphate  and  shake  vigorously 
to  insure  thorough  and  rapid  mixing  of  the  contents.  The  end 
point  should  be  sharper  than  in  standardizing  since  in  this  case 
there  is  no  green  color  due  to  chromium  and  the  solution  passes 
at  once  from  blue  to  nearly  colorless.  The  difference  between 
the  volume  of  thiosulphate  solution  required  for  the  blank  test 
and  that  required  for  the  solution  containing  the  sample  gives 
a  measure  of  the  iodine  absorbed  by  the  latter.  Calculate  the 
iodine  absorbed  in  terms  of  percentage  of  the  original  weight  of 
sample. 

Notes.  —  Most  of  the  difficulties  which  are  met  in  using  this 
method  are  due  to  impure  reagents  or  failure  to  observe  care- 
fully the  conditions  worked  out  by  Hiibl.  Probably  the  most 
important  sources  of  error  are :  (1)  The  use  of  impure  chloro- 
form or  water  containing  dissolved  air  causing  a  liberation  of 
iodine  from  the  potassium  iodide,  or  the  iodine  addition  product 
of  the  oil;  (2)  loss  of  iodine  from  the  use  of  vessel  with  im- 
perfectly fitting  stopper  or  from  titrating  at  too  high  tempera- 
ture or  with  too  much  exposure  to  air;  (3)  deterioration  of  the 
iodine  solutions  due  chiefly  to  impurities  in  the  alcohol  or 
too  high  temperature;  (4)  insufficient  excess  of  iodine  over 
that  absorbed;  (5)  variations  in  the  length  of  time  allowed  for 
the  reaction. 

The  small  amount  of  oil  needed  for  this  determination  can  con- 
veniently be  drawn  from  near  the  center  of  the  sample  bottle 
by  means  of  clean  glass  tubing  of  about  2  mm.  internal  diameter. 
Use  a  piece  of  thin  tubing  like  a  pipette,  wiping  it  free  from  oil 
on  the  outside  and  allowing  it  to  deliver  drop  by  drop  into  the 


152  METHODS   OF   ORGANIC    ANALYSIS 

weighed  flask  which  should  then  be  stoppered  and  reweighed  at 
once.  If  more  than  one  sample  of  oil  is  to  be  tested,  delays  can 
be  avoided  by  having  a  number  of  pieces  of  the  tubing  cleaned 
and  dried  in  advance  so  that  each  can  be  rejected  after  using  it 
for  one  sample. 

The  best  results  are  obtained  by  using  such  proportions  of  oil 
and  of  iodine  solution  as  to  have  present  from  two  to  three 
times  the  amount  of  iodine  which  will  be  absorbed.  The  form 
of  vessel  to  be  used  for  the  test  is,  of  course,  immaterial  so  long 
as  loss  of  iodine  is  avoided.  Some  prefer  to  weigh  the  sample 
on  a  small  watch  glass  and  place  the  latter  with  the  sample  in 
a  wide-mouth  glass  stoppered  bottle.  A  blank  test  must  be 
made  with  each  determination,  or  if  several  samples  are  treated 
at  once,  there  should  be  at  least  two  or  three  blanks. 

For  discussion  of  the  theory  of  the  action  of  Hiibl's  solution 
see  Lewkowitsch's  Oils,  Fats,  and  Waxes,  or  Gill's  Oil  Analysis. 

Method  of  Wijs 

Wijs  found  that  a  solution  of  iodine  monochloride  in  glacial 
acetic  acid  acts  in  the  same  manner  as  Hiibl's  solution,  but 
more  quickly.  Moreover,  the  reagent  is  much  more  stable 
than  that  of  Hiibl,  so  that  the  same  solution  can  be  used  for 
several  months  and  blank  tests  are  not  so  frequently  required. 

Reagents. — 1.  Glacial  acetic  acid.  This  must  contain  not 
over  0.5  per  cent  of  water  and  no  impurity  capable  of  reducing 
potassium  dichromate.  Test  by  adding  a  small  amount  of  a 
solution  of  dichromate  in  sulphuric  acid  and  warming  on  a 
water  bath ;  no  green  tinge  should  appear. 

2.  Iodine  chloride  solution.  Dissolve  12.5  to  13  grams  of 
iodine  in  a  liter  of  the  glacial  acetic  acid,  warming  gently  to 
aid  solution,  if  necessary.  Cool,  withdraw  25  cc.,  add  potas- 
sium iodide  solution,  and  determine  the  iodine  content  by  titra- 
tion  with  standard  thiosulphate ;  into  the  remainder  of  the 
solution  pass  a  current  of  chlorine  until  the  deep  iodine  color 
changes  to  an  orange  yellow,  showing  that  the  conversion  of 
iodine  to  iodine  monochloride  is  complete.  A  portion  of  the 


OILS,    FATS,    AND   WAXES  153 

solution  titrated  with  thiosulphate  should  now  show  twice  the 
original  halogen  content.  In  case  an  excess  of  chlorine  is 
found,  a  weighed  amount  of  iodine  equivalent  to  this  excess 
should  be  dissolved  in  the  solution. 

3.  Standard    solution   of    sodium    thiosulphate,    as   in   the 
method  of  Hiibl. 

4.  Pure  chloroform,  potassium  iodide,  starch  solution,  and 
distilled  water,  as  described  under  Hiibl's  method. 

Determination.  —  The  determination  is  carried  out  in  exactly 
the  manner  described  for  the  Hiibl  method  except  that  10  cc. 
of  the  potassium  iodide  solution  are  sufficient  and  a  much 
shorter  time  is  required  for  the  reaction.  With  non-drying 
oils  the  addition  of  iodine  chloride  is  usually  complete  in  15 
minutes.  Ordinarily  the  solution  should  be  titrated  after 
standing  one  half  hour. 

Notes.  —  As  the  solution  of  iodine  chloride  in  acetic  acid  is 
more  viscous  and  has  a  higher  coefficient  of  expansion  than 
ordinary  aqueous  solutions,  the  portions  added  must  be  meas- 
ured as  carefully  as  possible,  allowing  a  uniform  time  for  the 
burette  to  drain,  and  observing  that  the  temperature  does  not 
vary  more  than  three  or  four  degrees.  With  these  precautions 
it  is  not  always  necessary  to  make  a  blank  test  with  each 
determination  after  a  sufficient  number  of  such  tests  have  been 
made  to  show  that  the  solution  is  not  changing  appreciably  in 
strength. 

Method  of  Hanus 

Hanus  suggested  that  Wijs'  method  be  modified  by  the  use 
of  iodine  bromide  in  place  of  iodine  chloride.  As  an  excess  of 
iodine  does  no  harm,  the  preparation  of  the  reagent  is  simpler 
than  in  the  preceding  method. 

The  details  of  this  method  as  adopted  by  the  Association  of 
Official  Agricultural  Chemists  in  1905  are  essentially  as  fol- 
lows : 

Reagents. — 1.  Iodine  solution.  —  (a)  Dissolve  13.2  grams 
iodine  in  1000  cc.  glacial  acetic  (99.5  per  cent)  acid  (showing 
no  reduction  with  bichromate  and  sulphuric  acid),  add  enough 


154  METHODS  OF  ORGANIC  ANALYSIS 

bromine  to  double  the  halogen  content  determined  by  titra- 
tion  ;  3  cc.  of  bromine  is  about  the  proper  amount.  The 
iodine  may  be  dissolved  by  the  aid  of  heat,  but  the  solution 
should  be  cold  when  bromine  is  added. 

2.  Decinormal    sodium     thiosulphate    solution.  —  Dissolve 
24.8   grains   of  chemically   pure  sodium   thiosulphate,   freshly 
pulverized  as  finely  as  possible,  and  dried  between  filter  or  blot- 
ting paper  and  dilute  with  water  to  1  liter  at  the  temperature 
at  which  the  titrations  are  to  be  made.      [This  solution  is  to  be 
standardized  when  used  as  described  below.] 

3.  Starch  paste.  —  One  gram  of  starch  is  boiled  in  200  cc. 
of  distilled  water  for  10  minutes  and  cooled  to  room  tempera- 
ture. 

4.  Solution  of  potassium  iodide.  —  One  hundred  and  fifty 
grams  of  potassium  iodide  are  dissolved  in  water  and  made  up 
to  1  liter. 

5.  Decinormal    potassium    dichromate.  —  Dissolve    4.9066 
grams   of   chemically  pure  potassium   dichromate  in   distilled 
water,  and  make  the  volume  up  to  1  liter  at  the  temperature 
at  which  the  titrations  are  to  be  made.     The  dichromate  solu- 
tion should  be  checked  against  pure  iron. 

Determination.  —  (1)  Standardizing  the  sodium  thiosulphate 
solution. — Place  20  cc.  of  the  potassium  dichromate  solution, 
to  which  has  been  added  10  cc.  of  the  solution  of  potassium 
iodide,  in  a  glass-stoppered  flask.  Add  to  this  5  cc.  of  strong 
hydrochloric  acid.  Allow  the  solution  of  sodium  thiosulphate 
to  flow  slowly  into  the  flask  until  the  yellow  color  of  the  liquid 
has  almost  disappeared.  Add  a  few  drops  of  the  starch  paste, 
and  with  constant  shaking  continue  to  add  the  sodium  thiosul- 
phate solution  until  the  blue  color  just  disappears. 

(2)  Weighing  the  sample.  —  Weigh  about  one  half  gram  of 
fat,  0.25  gram  of  salad  oil,  or  0.10-0.20  gram  of  drying  oil  on 
a  small  watch  crystal  and  introduce  the  watch  crystal  into  a  wide- 
mouth  16-ounce  bottle  with  ground-glass  stopper  [or  weigh  the 
portion  in  a  glass-stoppered  Erlenmeyer  flask  as  described  under 
the  Hiibl  method  above]. 

(3)  Absorption  of  iodine.  —  The  fat  or  oil  in  the  bottle  is 


OILS,    FATS,    AND    WAXES  155 

dissolved  in  10  cc.  of  chloroform.  After  complete  solution  has 
taken  place,  25  cc.  of  the  iodine  solution  are  added.  Allow  to 
stand,  with  occasional  shaking,  for  thirty  minutes.  The  excess 
of  iodine  should  be  at  least  60  per  cent  of  the  amount  added. 

(4)  Titration  of  the  unabsorbed  iodine. — Add  10  cc.  of  the 
potassium  iodide  solution  arid  shake  thoroughly,  then  add  100 
cc.  of  distilled  water  to  the  contents  of  the  bottle.     Titrate  the 
excess  of  iodine  with  the  sodium  thiosulphate  solution,  which  is 
added  gradually,  with  constant  shaking,  until  the  yellow  color 
of  the  solution  has  almost  disappeared.     Add  a  few  drops  of 
starch  paste,  and  continue  the  titration  until  the  blue  color  has 
entirely  disappeared.     Toward  the  end  of  the  reaction  stopper 
the  bottle  and  shake  violently,  so  that  any  iodine  remaining  in 
solution  in  the  chloroform  may  be  taken  up  by  the  potassium 
iodide  solution. 

(5)  Setting  the  value  of  iodine  solution  by  blank  determina- 
tions.— At  the  time  of  adding  the  iodine  solution  to  the  fat  run 
the  same  amount  into  each  of  two  bottles  of  the  same  size  as 
those    used    for   the  determination  and  carry  these  "  blanks " 
through  exactly  the  same  manipulation  as  in  the  determination 
upon  the  fat.     These  blank  experiments  must  be  made  each  time 
the  iodine  solution  is  used. 

Notes.  —  Great  care  must  be  taken  that  the  temperature  of  the 
solution  does  not  change  during  the  time  of  the  operation,  as 
acetic  acid  has  a  very  high  coefficient  of  expansion,  and  a  slight 
change  of  temperature  makes  an  appreciable  difference  in  the 
strength  of  the  solution.  Where  any  great  number  of  determina- 
tions are  to  be  made,  blanks  should  be  measured  out  at  short 
intervals.  This  precaution  applies  as  well  to  the  use  of  the  Hubl 
solution,  as  the  coefficient  of  the  expansion  of  alcohol  is  large. 

When  the  potassium  iodide  is  added,  the  solution  should  be 
thoroughly  mixed  before  the  addition  of  water. 

The  acetic  acid  must  be  full  strength  and  pure  in  order  to 
obtain  a  solution  which  will  keep  well. 

This  method  has  several  advantages  over  that  of  Hubl,  the 
most  important  being  that  the  reagent  keeps  better  and  acts  more 
rapidly  so  that  there  is  less  danger  of  discrepancies  from  sec- 


156  METHODS   OF   ORGANIC    ANALYSIS 

ondary  reactions.  In  general  the  apparatus  and  manipulations 
described  under  the  Hiibl  method  apply  also  here  and  should  be 
carefully  noted.  In  this  case  the  flasks  may  be  sealed  by  pouring 
a  little  glacial  acetic  acid  into  the  gutter  instead  of  the  potassium 
iodide  described  under  the  Hiibl  method,  or  since  the  time  of 
standing  is  here  only  30  minutes  an  exceptionally  well-ground 
stopper  may  not  require  any  liquid  seal  to  prevent  the  escape  of 
iodine. 

Comparison  of  the  Hubl,  Wijs,  and  If  anus  Methods 

Wijs  based  his  modification  on  the  belief  that  iodine  chloride 
is  the  active  substance  in  Hiibl's  solution  and  that  the  more 
stable  solution  of  iodine  chloride  in  acetic  acid  should  give  the 
same  theoretical  results  with  greater  certainty.  That  higher 
results  were  often  obtained  by  his  method  Wijs  attributed  to 
the  deterioration  or  incomplete  action  of  the  Hiibl  solution  as 
ordinarily  prepared.  Lewkowitsch1  confirms  this,  stating  that, 
when  the  Hiibl  method  is  carried  out  under  the  best  conditions, 
the  results  of  the  two  methods  agree. 

Tolman  and  Munson,2  however,  in  an  extended  comparison 
of  the  three  methods  almost  invariably  obtained  higher  results 
by  the  Wijs  than  by  the  Hiibl  method.  Hanus'  method  gave 
intermediate  results,  usually  agreeing  quite  closely  with  the 
Hiibl  numbers.  Similar  results  on  a  smaller  number  of  samples 
had  previously  been  published  by  Hunt3  and  have  since  been 
obtained  by  W.  G.  Tice.4  For  oils  having  iodine  numbers  be- 
low 100,  the  three  methods  can  be  used  interchangeably,  as  the 
differences  in  results  will  rarely  exceed  1.5  units,  which  is  only 
about  one  tenth  of  the  variation  which  occurs  among  pure  oils 
of  the  same  species.  In  applying  the  Wijs  solution  to  oils  hav- 
ing much  higher  iodine  numbers  it  is  to  be  remembered  that 
results  as  much  as  10  units  in  excess  of  these  given  by  Hiibl's 
solution  will  sometimes  be  obtained. 

The  method  of  Hiibl  is  "  official  "  in  the  United  States,  but 

1  Analyst,  1899,  24,  259.  2  J.  Amer.  Chem.  Soc.,  1903,  25,  244. 

3  J.  Soc.  Chem.  Ind..  1902,  21,  454. 

4  In  the  Havemeyer  Laboratory,  Columbia  University. 


OILS,    FATS,    AND    WAXES  157 

that  of  Hanus  lias  been  adopted  by  the  Association  of  Official 
Agricultural  Chemists  as  an  optional  method  for  the  examina- 
tion of  edible  oils  and  fats.  The  method  of  Wijs  is  more  com- 
monly used  in  England  and  is  recommended  in  preference  to  the 
Hanus  method  by  Lewkowitsch 1  and  Archbutt,2  the  latter  point- 
ing out  especially  that  the  Wijs  solution  gives  approximately  the 
theoretical  iodine  number  for  turpentine  (which  is  often  mixed 
with  drying  oils)  while  that  of  Hanus  gives  much  lower  results. 
In  comparing  analytical  results  with  the  numbers  found  .in 
"  tables  of  constants  "  it  should  be  remembered  that  the  latter 
are  based  in  part  upon  results  obtained  (not  always  under  the 
best  conditions)  by  Hubl's  method,  and  in  part  upon  later  re- 
sults determined  according  to  Wijs.  Such  tables  tend  to  show 
a  greater  range  than  would  be  found  by  the  use  of  either  method 
alone,  this  being  especially  true  of  the  oils  having  high  iodine 
numbers. 

MAUMENE  NUMBER  —  SPECIFIC  TEMPERATURE  REACTION 

Long  before  the  introduction  of  any  of  the  halogen  absorp- 
tion methods,  Maumene  3  tested  olive  oil  for  adulterants  such  as 
poppyseed  oil  by  mixing  fixed  volumes  of  oil  with  strong  sul- 
phuric acid  and  observing  the  rise  in  temperature  which  is  much 
greater  with  seed  oils  than  with  olive.  The  rise  in  temperature, 
expressed  in  degrees  C.,  which  occurs  on  mixing  10  cc.  of  strong 
sulphuric  acid  with  50  grams  of  oil  is  commonly  known  as  the 
Maumene  number.  Thomson  and  Ballantyne  4  pointed  out  the 
discrepancies  which  may  be  introduced  through  variations  in  the 
strength  of  the  acid  used,  and  showed  that  they  can  be  largely 
avoided  by  comparing  the  rise  observed  in  the  case  of  an  oil 
with  that  shown  by  water  under  the  same  conditions,  the  latter 
being  taken  as  100.  The  result  thus  obtained  is  known  as  the 
specific  temperature  reaction  or  specific  Maumene  number. 
Thus  if  50  grams  of  water  mixed  with  10  cc.  of  sulphuric  acid 
showed  a  rise  of  40°  and  50  grams  of  an  oil  under  the  same  con- 

1  Oils,  Fats,  and  Waxes  (4th  Ed.),  I.,  323. 

2  J.  Soc.  Chem.  Ind.,  1904,  23,  306.  3  Compt.  rend.,  1852,  35,  572. 
4  J.  Soc.  Chem.  Ind.,  1891,  10,  234. 


158  METHODS    OF   ORGANIC    ANALYSIS 

ditions  a  rise  of  36°,  the  specific  Maumene  number  of  the  oil 
would  be  90. 

With  fresh  fatty  oils  the  rise  of  temperature  depends  mainly 
upon  the  presence  of  glycerides  of  the  unsaturated  acids,  and 
the  Maumene  and  Hiibl  numbers  are  therefore  roughly  propor- 
tional. This  relation,  however,  is  not  sufficient  to  permit  of 
the  determination  of  the  Maumene  in  place  of  the  iodine  num- 
ber except  for  rough  work  or  as  a  preliminary  test.  The 
greatest  value  of  this  temperature  reaction  lies  in  the  fact  that 
as  oils  become  altered  by  absorption  of  oxygen  from  the  air, 
the  Maumene  numbers  increase  while  the  iodine  numbers  de- 
crease. The  significance  of  this  relation  is  more  fully  ex- 
plained in  Chapter  X. 

Apparatus  and  Reagents.  —  A  deep  beaker  of  150  t6  200  cc. 
capacity  should  be  used  for  this  test  and  should  be  so  jacketed 
as  to  prevent  a  rapid  loss  of  heat.  This  is  done  by  placing  it 
in  a  larger  beaker  or  a  metal  cup  and  filling  the  space  with 
asbestos  or  other  dry  porous  material.  If  a  new  nest  of 
beakers  is  at  hand,  the  test  can  be  made  in  one  of  the  smaller 
beakers  using  the  remainder  of  the  nest  with  the  packing 
intact  as  a  jacket.  The  insulation  should  be  sufficient  to  pre- 
vent the  outer  vessel  from  becoming  perceptibly  warm  when 
a  test  is  made.  The  only  reagent  required  is  strong  sulphuric 
acid  which  can  be  added  either  from  a  burette  or  (more  satis- 
factorily) from  a  10-cc.  pipette.  For  observing  the  rise  in 
temperature  use  a  thermometer  graduated  in  degrees  on  which 
the  readings  can  be  taken  with  accuracy  to  0.2  degree. 

Determination.  —  The  oils  to  be  tested,  .the  acid  and  appa- 
ratus to  be  used,  and  the  distilled  water  which  is  to  serve  for 
comparison  must  all  have  the  same  initial  temperature,  which 
should  be  between  20°  and  24°.  This  will  usually  be  the  case 
if  they  have  stood  side  by  side  at  room  temperature  for  several 
hours.  Measure  50  cc.  of  water  into  the  beaker,  introduce  the 
thermometer,  and  note  the  temperature;  add  10  cc.  of  the  acid, 
stirring  thoroughly  with  the  thermometer  so  that  the  solution 
is  kept  well  mixed  and  the  temperature  rises  steadily.  At 
intervals  of  a  few  seconds  bring  the  bulb  of  the  thermometer 


OILS,    FATS,    AND   WAXES  159 

to  the  center  of  the  solution  and  observe  the  temperature. 
Record  the  highest  reading  found.  When  the  mixture  in  the 
beaker  has  reached  its  maximum  temperature,  it  should  be  re- 
jected at  once  to  avoid  unnecessary  warming  of  the  apparatus, 
since  the  initial  temperature  must  be  restored  before  beginning 
another  test.  After  the  rise  with  water  has  been  found  by 
duplicate  determinations  which  do  not  differ  by  more  than 
0.4°,  dry  the  beaker  thoroughly,  weigh  into  it  50  grams  of  oil 
(within  0.05  gram),  and  treat  with  10  cc.  of  acid  in  exactly 
the  same  manner.  The  mixture  often  becomes  very  viscous, 
requiring  vigorous  stirring  to  insure  a  uniform  temperature. 
In  reporting  results  give  the  actual  rise  observed  with  oil  and 
with  water  as  well  as  the  specific  temperature  reaction. 

Notes.  —  By  the  use  of  a  pipette  having  a  fine  outlet  requir- 
ing 30  to  60  seconds  for  the  10  cc.  of  acid  to  flow  into  the 
beaker,  more  thorough  mixing  becomes  possible  and  better  re- 
sults are  secured.  In  order  to  avoid  trouble  in  cleaning  the 
beaker  it  should  be  emptied  and  wiped  with  dry  cotton  waste 
or  porous  paper  while  still  warm.  The  acid  used  must  be  pro- 
tected from  unnecessary  exposure  to  air,  as  it  readily  absorbs 
moisture  to  an  extent  sufficient  to  cause  an  appreciably  lower 
temperature  reaction.  In  testing  edible  and  lubricating  oils 
the  strongest  available  sulphuric  acid  should  be  used.  In  the 
case  of  drying  oils  and  fish  or  other  marine  animal  oils,  the  ad- 
dition of  such  strong  acid  to  the  undiluted  oil  causes  violent 
frothing  and  decomposition.  This  may  be  avoided  1  by  the  use 
of  sulphuric  acid  of  such  strength  as  to  give  a  rise  of  35°  when 
added  to  water  under  the  conditions  to  be  used  in  testing  the 
oil.  An  important  modification  of  this  test  has  been  proposed 
by  Mitchell.2 

In  mixtures,  especially  of  fatty  with  mineral  oils,  this  temper- 
ature reaction  is  not  to  be  relied  upon  as  an  additive  property. 
Sherman,  Danziger,  and  Kohnstamm3  found  that  mixtures  of 

1  Sherman,  Danziger,  and  Kohnstamm:  J.  Am.  Chem.  Soc.,  1902,  24,  266. 

2  Analyst,  1901,  26,  169.     See  also  Sherman  and  Falk :  J.  Am.  Chem.  Soc., 
1903,  25,  713. 

*J.  Am.  Chem.  Soc.,  24,  269. 


160  METHODS   OF   ORGANIC    ANALYSIS 

various  fatty  oils  with  mineral  oil  gave  results  much  higher 
than  would  correspond  to  the  known  constituents  of  the  mix- 
ture. Similar  results  were  afterward  published  by  Suzzi,1  and 
later  Richter2  experimented  with  mixtures  of  fatty  oils  and 
reported  that  the  addition  of  5  to  10  per  cent  of  sunflower, 
rape,  or  peanut  oil  depressed  the  specific  temperature  reaction  of 
olive  oil,  and  other  mixtures  of  olive  and  rape  or  olive  and  pea- 
nut oils  gave  results  constantly  lower  than  the  calculated 
values,  while  with  mixtures  of  olive  and  sunflower  oils  contain- 
ing 20  per  cent  or  more  of  the  latter  the  results  found  were 
sometimes  above  and  sometimes  below  the  calculated  values, 
the  differences  being  never  greater  than  5  units. 

The  question  was  then  reinvestigated  by  Boynton  and  Sher- 
man,3 using  an  acid  which  gave  with  water  a  rise  of  44°  C.  and  car- 
rying out  the  tests  as  described  above.  In  a  series  of  tests  upon 
40  samples  (7  individual  oils  and  33  mixtures)  it  was  found 
that  the  specific  temperature  reactions  of  mixtures  of  fatty  oils 
did  not  as  a  rule  differ  greatly  from  the  values  calculated  on  the 
assumption  that  this  is  an  additive  property.  Where  differences 
were  found,  the  observed  temperature  reaction  of  the  mixture 
of  two  fatty  oils  was  always  lower  than  the  calculated  value. 
On  the  other  hand,  in  mixtures  of  fatty  and  mineral  oils  the 
values  found  were  invariably  higher  than  those  calculated,  the 
difference  between  the  theoretical  and  the  observed  values 
apparently  depending  more  upon  the  proportion  of  mineral  oil 
than  upon  the  nature  of  the  fatty  oil  present. 

More  recently,  Kessler  and  Mathiason4  have  also  reported 
that  the  Maumene  test  is  not  an  additive  property. 


THE  ACETYL  NUMBER 

The  acetyl  number  is  the  number  of  milligrams  of  potassium 
hydroxide  required  to  neutralize  the  acetic  acid  obtained  by 
saponification  of  one  gram  of  the  acetylated  fat  or  wax. 

lBoll.  chim.  farm.,  44,  301 ;  Chem.  ZentrU.,  1905,  II,  80. 

2Z.  angew.  Chem.,  1907,  1605. 

3  School  of  Mines  Quarterly,  31,  64.  4  J.  Ind.  Eng.  Chem.,  3,  66. 


OILS,    FATS,    AND   WAXES  161 

It  indicates  the  proportion  of  hydroxyl  groups  in  the  original 
substance.  If  the  latter  consists  essentially  of  triglycerides, 
the  acetyl  number  serves  as  a  measure  of  the  hydroxy-acids 
present.  Free  alcohols,  if  present,  increase  the  acetyl  numbers. 
Lewkowitsch  finds  that  free  fatty  acids  of  the  stearic  series 
may  also  react  in  such  a  way  as  to  increase  the  apparent  acetyl 
numbers,  and  therefore  prefers  to  acetylate  the  original  fat  or 
wax  rather  than  to  work  with  the  mixed  fatty  acids  as  was 
previously  recommended  by  Benedikt  and  Ulzer.1 

The  method  of  Lewkowitsch  is  essentially  as  follows : 2 

Boil  10  grams  of  the  substance  with  twice  its  weight  of  acetic 
anhydride  in  a  round-bottomed  flask  under  a  reflux  condenser 
for  two  hours;  pour  the  resulting  mixture  into  a  large  beaker 
containing  500  cc.  of  hot  water  and  boil  for  half  an  hour,  passing 
a  slow  current  of  carbon  dioxide  through  the  solution  to  pre- 
vent bumping.  Allow  the  mixture  to  separate  into  two  layers, 
siphon  off  the  water,  and  boil  the  oily  layer  with  three  successive 
portions  of  fresh  water,  the  last  of  which  should  not  react  acid 
to  litmus  paper. 

All  free  acetic  acid  having  been  removed,  the  acetylated  fat 
is  carefully  separated  from  water  and  further  dried  by  filtering 
through  anhydrous  paper  in  a  drying  oven. 

Weigh  2  to  5  grams  of  the  acetylated  fat  and  saponify  with 
a  measured  volume  of  standard  alcoholic  potash  as  in  the  deter- 
mination of  the  saponification  number;  evaporate  nearly  to 
dryness  to  expel  the  alcohol,  dissolve  the  soap  in  water,  and  add 
an  amount  of  standard  sulphuric  acid  exactly  equivalent  to  the 
alkali  used  for  saponification.  Warm  gently  until  the  fatty 
acids  separate  as  a  layer  at  the  top.  Filter  through  wet  paper, 
wash  the  fatty  acids  with  boiling  water  until  the  filtrate  is  no 
longer  acid,  and  titrate  the  filtrate  and  washings  with  tenth- 
normal  alkali,  using  phenolphthalein  as  indicator.  Deduct  the 
amount  of  alkali  required  to  neutralize  any  soluble  fatty  acids 
in  the  original  substance  and  calculate  the  acetic  acid  found,  in 
terms  of  acetyl  number,  as  defined  above. 

Instead  of  filtering  and  washing  with  water,  the  acetic  acid 

iMonatsh.  Chem.,  1887,  8,  40.  2<7.  Soc.  Chem.  Ind.,  1897,  16,  503. 


162  METHODS   OF   ORGANIC   ANALYSIS 

can  be  separated  from  the  fatty  acids  by  distilling  with  steam, 
but  this  requires  considerably  more  time  than  the  nitration 
process. 

In  the  examination  of  waxes  the  acetyl  numbers  show  the 
proportional  amounts  of  free  alcohols.  Fats  and  fatty  oils  con- 
tain free  alcohols  (cholesterol,  phytosterol,  etc.),  but  in  very 
small  amounts  only,  so  that  the  acetyl  numbers  are  low  except 
in  oils  which  contain  hydroxy-acids  (Chapter  VII).  The 
acetyl  number  of  castor  oil  is  about  150.  Other  oils  rarely 
show  acetyl  numbers  higher  than  10  or  15,  unless  they  have 
been  exposed  to  atmospheric  oxidation,  in  which  case  hydroxy- 
acids  may  have  been  formed  from  the  unsaturated  acids  orig- 
inally present. 

SPECIFIC  GRAVITY 

The  specific  gravities  of  the  glycerides  depend  upon  the  fatty 
acids  which  they  contain.  Comparing  acids  of  the  same  homol- 
ogous series  the  specific  gravity  was  found  to  decrease  with  in- 
creasing molecular  weight;  while  between  corresponding  acids 
of  different  series  it  increases  with  the  number  of  double  bonds 
and  of  hydroxyl  groups. 

Fresh  specimens  of  the  same  kind  vary  but  little  in  specific 
gravity,  and  the  determination  of  this  property  is  often  useful 
in  differentiating  oils,  fats,  and  waxes,  and  in  testing  them  for 
adulterations.  The  determination  can  be  made  either  by  means 
of  a  very  delicate  special  hydrometer,  by  the  Westphal  balance 
(Fig.  11),  or  by  one  of  the  methods  described  in  Chapter  I. 
On  account  of  the  high  coefficient  of  expansion  of  oils  it  is  im- 
portant that  the  temperature  at  which  the  specific  gravity  is 
taken  be  accurately  known.  This  is  conveniently  accomplished 
by  means  of  a  hydrometer  having  a  thermometer  scale  in  the 
stem,  or  by  using  the  thermometer  sinker  with  the  Westphal 
balance. 

Directions  for  the  use  of  the  Westphal  balance  are  usually 
supplied  with  the  instrument.  It  should  always  be  placed 
upon  a  firm  level  table  and  very  carefully  adjusted  before  using. 
In  setting  up  the  instrument  be  sure  that  the  leveling  screw  is 


OILS,    FATS,    AND    WAXES 


163 


directly  beneath  the  arm  which  supports  the  beam.  Place  the 
latter  in  position  and  hang  the  sinker.  Compare  the  height  of 
the  beam  with  that  of  the  cylinder  which  is  to  contain  the  oil, 
and  lengthen  or  shorten  the  standard  if  necessary,  then  carefully 
adjust  the  balance  by  means  of  the  leveling  screw  until  the 
point  projecting  from  the  end  of  the  beam  stands  exactly  op- 
posite the  fixed  point  at  the  left.  Nearly  fill  the  cylinder  with 
the  oil  at  about  14°.  Lift  the  sinker,  place  the  cylinder  under 


FIG.  11.  —The  Westphal  balance. 

the  end  of  the  beam,  and  replace  the  sinker  so  that  it  hangs 
freely  in  the  oil.  The  specific  gravity  is  now  found  by  placing 
weights  on  the  beam  until  the  balance  is  restored.  Make  the 
final  adjustment  of  the  smallest  weight  when  the  thermometer 
in  the  sinker  shows  the  desired  temperature —for  oils,  15.5°. 
The  specific  gravity  is  read  directly  from  the  position  of  the 
weights  on  the  beam.  If  the  largest  weight  is  at  9,  the  second 
at  3,  the  third  at  1,  and  the  fourth  at  4,  the  specific  gravity  of 
the  oil  is  0.9314.  The  readings  should  be  made  to  four  decimal 
places  (i.e.  all  four  sizes  of  weights  should  be  used)  and  the 


164  METHODS  OF   ORGANIC   ANALYSIS 

error  of  determination  should  not  exceed  0.0005.  By  using 
the  same  sinker  on  the  analytical  balance  or  by  weighing  the 
oil  in  a  pyknometer,  more  accurate  results  can  be  obtained. 
The  Westphal  balance,  is,  however,  sufficiently  delicate  for 
routine  work  if  carefully  used.  Care  must  be  taken  that  the 
sinker  is  always  suspended  at  the  same  depth  in  the  liquid 
under  examination.  The  graduation  of  the  beam  should  be 
tested  to  see  that  the  spaces  are  all  of  equal  length,  and  the  re- 
sults should  occasionally  be  verified  by  comparative  determina- 
tions with  the  pykometer.  In  working  with  very  viscous  oils 
it  is  advantageous  to  use  a  sinker  of  high  specific  gravity.1 

If  a  delicate  form  of  Westphal  balance  is  not  at  hand  or  if 
more  accurate  results  are  desired,  the  pyknometer  will  be  found 
well  suited  to  this  work. 

For  calculating  data  obtained  at  higher  temperatures  to  the 
standard  temperature  of  15.5°  C.,  Wright2  gives  the  following 
factors  :  16°,  1.00035  ;  17°,  1.00106  ;  18°,  1.00177  ;  19, 1.00248; 
20°,  1.00319;  21°,  1.00391;  22°,  1.00462;  23°,  1.00534;  24°, 
1.00605  ;  25°,  1.00677.  Since  the  different  oils  vary  somewhat 
in  coefficient  of  expansion  it  is  better  to  work  at  the  standard 
temperature  than  to  depend  upon  factors  for  correcting  results. 

For  the  determination  of  specific  gravity  at  temperatures  con- 
siderably above  that  of  the  laboratory  it  is  convenient  to  use  an 
Ostwald  pyknometer.  This  is  filled  with  the  sample,  suspended 
in  water  at  the  required  temperature,  and  adjusted,  then  removed, 
dried  on  the  outside,  cooled  to  room  temperature,  and  weighed. 
For  a  detailed  description  of  the  standardization  of  pyknometers 
at  high  temperatures  consult  any  standard  work  on  physico- 
chemical  measurements  or  Bulletin  107,  Bureau  of  Chemistry, 
U.  S.  Department  of  Agriculture. 

INDEX  OF  REFRACTION 

The  index  of  refraction,  like  the  specific  gravity,  increases 
with  the  proportion  of  unsaturated,  or  of  hydroxy,  acids.  Un- 

1  MeGill  :  Analyst,  1896,  21,  156. 

2  J.  Soc.  Chem.  2nd.,  1907,  26,  513.  Allen's  Commercial  Organic  Analysis, 
Fourth  Edition,  Vol.  II,  p.  51. 


OILS,    FATS,    AND   WAXES 


165 


like  the  specific  gravity,  the  index  of  refraction  increases  with 
the  molecular  weight  in  the  homologous  series  of  saturated  acids, 
so  that  the  presence  of  the  fatty  acids  of  low  molecular  weight 
in  butter  causes  this  fat  to  have  a  lower  index  of  refraction  than 
other  animal  fats,  while  its  specific  gravity  is  higher.  In  al- 
most all  of  the  fatty  oils  the  index  of  refraction  varies  with  the 
specific  gravity,  so  that  in  routine  work  it  is  not  necessary  to 
make  both  determinations,  but  either  may  be  used  to  confirm  the 
inferences  drawn  from  the  other  determinations.1 

The  index  of  refraction  has  usually  been  determined  by  means 
of  the  Abbe  or  the  Pulfrich  refractometer  or  the  "butyro-re- 
fractometer "    of    Zeiss. 
The  latter  has  an  arbi- 
trary   scale     of     known 
value     whose     readings 
may   be   converted    into 
actual    index    of   refrac- 
tion by  the  use  of   the 
table  on  page  166. 

The  construction  of 
the  butyro-refractometer 
is  shown  in  Fig.  12.  To 
take  the  index  of  refrac- 
tion of  an  oil,  tilt  the  in- 
strument until  the  glass 
plate  on  the  inner  sur- 
face of  B  is  horizontal, 
place  a  few  drops  of  the 
oil  upon  it,  then  close  it 
against  A  and  clamp  in 
position  by  turning  F. 
The  space  between  A  and  B  is  now  filled  with  oil,  and  by 
looking  through  the  telescope  and  tilting  the  mirror  J  until 
the  scale  is  well  illuminated,  one  obtains  a  measure  of  the  index 
of  refraction  of  the  oil  from  the  position  of  the  shadow  upon 

1  For  a  very  full  discussion  of  the  relation  of  index  of  refraction  to  specific 
gravity  in  oils  and  fats  see  Proctor:  J.  Soc.  Chem.  Ind.,  1898,  17,  1021. 


FIG.  12.  — The  Zeiss  butyro-refractometer. 


166 


METHODS  OF  ORGANIC  ANALYSIS 


the  scale.  Detailed  directions  for  the  care  and  adjustment  of 
the  instrument  are  furnished  by  the  manufacturers  and  may 
also  be  found  in  Leach's  Food  Inspection  and  Analysis. 


TABLE  14.  —  INDEX  OF  REFRACTION  CORRESPONDING  TO  SCALE  READING 
OF  BUTYRO-REFRACTOMETER 


Scale 
reading 

Index  of 
refraction 

Scale 
reading 

Index  of 
refraction 

Scale 
reading 

Index  of 
refraction 

Scale 
reading 

Index  of 
refraction 

0 

1.4220 

26 

1.4423 

51 

1.4600 

76 

1.4760 

1 

1.4228 

27 

1.4430 

52 

1.4607 

77 

1.4766 

2 

1.4236 

28 

1.4438 

53 

1.4613 

78 

1.4772 

3 

1.4244 

29 

1.4445 

54 

1.4620 

79 

1.4778 

4 

1.4252 

30 

1.4452 

55 

1.4626 

80 

1.4783 

5 

1.4260 

31 

1.4460 

56 

1.4633 

81 

1.4789 

6 

1.4268 

32 

1.4467 

57 

1.4640 

82 

1.4795 

7 

1.4276 

33 

1.4474 

58 

1.4646 

83 

1.4801 

8 

1.4284 

34 

1.4481 

59 

1.4653 

84 

1.4807 

9 

1.4292 

35 

1.4488 

60 

1.4659 

85 

1.4812 

10 

1.4300 

36 

1.4495 

61 

1.4666 

86 

1.4818 

11 

1.4308 

37 

1.4502 

62 

1.4672 

87 

1.4824 

12 

1.4316 

38 

1.4510 

63 

1.4679 

88 

1.4829 

13 

1.4324 

39 

1.4517 

64 

1.4685 

89 

1.4835 

14 

1.4331 

40 

1.4524 

65 

1.4691 

90 

1.4840 

15 

1.4339 

41 

1.4531 

66 

1.4698 

91 

1.4846 

16 

1.4347 

42 

1.4538 

67 

1.4704 

92 

1.4851 

17 

1.4354 

43 

1.4545 

68 

1.4710 

93 

1.4857 

18 

1.4362 

44 

1.4552 

69 

1.4717 

94 

1.4862 

19 

1.4370 

45 

1.4559 

70 

1.4723 

95 

1.4868 

20 

1.4377 

46 

1.4566 

71 

1.4729 

96 

1.4873 

21 

1.4385 

47 

1.4573 

72 

1.4736 

97 

1.4879 

22 

1.4392 

48 

1.4580 

73 

1.4742 

98 

1.4884 

23 

1.4400 

49 

1.4587 

74 

1.4748 

99 

1.4890 

24 

1.4408 

50 

1.4593 

75 

1.4754 

100 

1.4895 

25 

1.4415 

Since  the  butyro-refractometer  is  constructed  to  cover  only 
such  a  range  in  the  index  of  refraction  as  is  likely  to  be  met 
in  work  with  fats  and  oils,  it  is  possible  to  make  more  delicate 
readings  with  this  instrument  than  with  the  Abbe  refrac- 


OILS,    FATS,    AND    WAXES  167 

tometer.  It  is  preferable,  however,  to  express  all  results  in 
terms  of  the  index  of  refraction,  especially  as  some  substances 
frequently  mixed  with  oils  and  fats  are  too  highly  refractive  to 
be  examined  in  th*e  butyro-refractometer. 

The  index  of  refraction  decreases  with  rising  temperature. 
The  rate  of  change  is  nearly  constant  for  the  common  oils  and 
fats,  a  rise  of  1°  causing  a  diminution  of  0.000365  in  the  index 
of  refraction.1 

MELTING  AND  SOLIDIFYING  POINTS  —  TITEB  TEST 

The  melting  point  of  a  fat,  or  of  the  mixed  fatty  acids  obtained 
from  it,  increases  with  the  mean  molecular  weight  among  acids 
of  the  saturated  series,  while  in  mixtures  of  acids  of  the 
saturated  and  unsaturated  series  (or  their  glycerides)  the 
melting  point  becomes  lower  as  the  proportion  of  unsatu- 
rated compounds  increases.  The  melting  point  of  a  solid  fat 
is  best  determined  by  heating  a  thin  disk  of  the  substance 
suspended  in  a  mixture  of  water  and  alcohol  as  described 
in  Chapter  IX.  This  method  being  applicable  only  to  sub- 
stances practically  insoluble  in  alcohol,  the  melting  point 
of  a  mixture  of  fatty  acids  or  of  a  wax  may  be  taken  as 
follows  :  2 

Draw  up  the  melted  fatty  acids  (or  the  wax)  into  a  very 
thin-walled  capillary  tube  3  to  5  cm.  long  according  to  the 
length  of  the  bulb  of  the  thermometer  to  be  used.  Seal  one 
end  of  the  tube  and  allow  the  substance  to  cool  on  ice  for  12  to 
15  hours.  Attach  to  the  bulb  of  a  delicate  thermometer 
graduated  to  0.2°,  immerse  in  water,  and  heat  very  slowly. 
The  temperature  at  which  the  substance  becomes  transparent  is 
taken  as  the  melting  point. 

Natural  fats  sometimes  show  changeable  or  "  double  "  melt- 
ing points.  Griin  and  Schacht3  account  for  this  as  due  to 
the  presence  of  mixed  glycerides  capable  of  existence  in  two 

1  Proctor:   J.  Soc.  Chem.  Ind.,  1898,  17,  1023.     Tolman  and  Munson :  J. 
Am.  Chem.  Soc.,  1902,  24,  754. 

2  U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  107. 

3  JBer.,  1907,  40,  1778  ;  Allen  (4),  II,  52. 


168  METHODS   OF   ORGANIC   ANALYSIS 

isomeric  forms  of  different  melting  points,  one  of  which  forms 
may  be  gradually  changing  into  the  other. 

The  so-called  "  titer  test  "  is  the  determination  of  the  solid- 
ifying point  of  the  mixed  fatty  acids. 

VISCOSITY 

The  presence  of  glycerides  of  hydroxy-acids,  whether  natural, 
as  in  castor  oil,  or  artificial,  as  in  the  "  blown  "  oils  of  commerce, 
gives  a  very  high  viscosity  compared  with  fatty  oils  in  which 
hydroxy-acids  are  absent.  Among  the  latter,  viscosities  vary 
as  a  rule  with  the  melting  points  of  the  mixed  fatty  acids. 
While  the  viscosity  may  aid  in  the  identification  of  fats  and 
oils  in  certain  cases,  its  determination  is  especially  important 
in  the  examination  of  lubricating  oils  and  will,  therefore,  be  dis- 
cussed in  that  connection. 

HEAT  OF  COMBUSTION 

The  heat  of  combustion  of  fats  and  fatty  oils  is  a  property  as 
nearly  constant  as  the  specific  gravity  to  which  it  stands  in 
approximately  inverse  proportion,  being  lowered  by  the  pres- 
ence of  acids  of  low  molecular  weight,  highly  unsaturated 
acids,  or  acids  containing  hydroxyl  groups.  The  heats  of 
combustion  of  butter-fat,  drying  oils,  castor  oil,  and  blown  oils 
are,  therefore,  lower  than  those  of  body  fats  and  such  fatty  oils 
as  olive,  almond,  peanut,  or  rape-seed.  Waxes  and  hydrocar- 
bons have  higher  heats  of  combustion  than  the  glycerides.  The 
same  is  true  of  the  alcohols  of  very  high  molecular  weight,  such 
as  cholesterol  and  phytosterol  of  which  the  so-called  unsaponi- 
fiable  matter  of  fats  is  chiefly  composed.  With  a  suitable  oxy- 
gen calorimeter1  the  heat  of  combustion  can  be  very  accurately 
determined  and  the  results  are  often  useful  in  verifying  the 
conclusions  drawn  from  other  determinations.  For  fuller  dis- 
cussions see  J.  Am.  Chem.  Soc.,  1896,  18,  178;  1901,  23,  164; 
1902,  24,  348  ;  1903,  25,  659. 

1  For  discussion  of  calorimeters  and  determination  of  heat  of  combustion  see 
Chapter  XII. 


OILS,    FATS,    AND   WAXES  169 

ALCOHOLS  OF  FATS  AND  WAXES 

(UNSAPONIFIABLE  MATTER) 

Under  the  term  "  unsaponifiable  matter  "  are  included  all  sub- 
stances found  in  fats  and  waxes  which  are  insoluble  in  water 
and  do  not  combine  with  caustic  alkali  to  form  soluble  soaps. 
In  a  commercially  pure  fat  or  wax  the  "  unsaponifiable  matter" 
consists  chiefly  of  one  or  more  alcohols  of  high  molecular 
weight,  the  more  important  of  which  are  mentioned  below. 

The  common  waxes  yield  50  to  55  per  cent  of  insoluble  alco- 
hol, but  the  total  amount  of  unsaponifiable  matter  in  fats  is 
much  lower,  being  usually  less  than  1  per  cent  in  animal  and 
less  than  2  per  cent  in  vegetable  fats. 

ALCOHOLS  OF  THE  SERIES  CnH2n+2O 

Cetyl  alcohol,  C16H34O,  occurs  as  palmitate  in  spermaceti.  It 
is  a  tasteless  and  odorless  solid,  insoluble  in  water,  but  soluble 
in  alcohol  and  very  easily  soluble  in  ether  or  benzene.  Melting 
point  50°;  specific  gravity  at  99°,  0.783T. 

Octodecyl  alcohol,  C18H38O,  also  occurs  as  ester  in  spermaceti. 
It  is  similar  in  properties  to  cetyl  alcohol.  Melting  point  59° ; 
specific  gravity  at  99°,  0.7849. 

Myricyl  alcohol,  C30H62O,  occurs  as  palmitate  in  beeswax  and 
both  free  and  combined  in  carnaiiba  wax.  It  melts  at  85°  to 
88°  and  is  nearly  insoluble  in  cold,  but  readily  soluble  in  hot, 
alcohol. 

Other  alcohols  of  this  series  are  known,  but  need  not  be  con- 
sidered here.  From  the  examples  given  it  will  be  seen  that  the 
melting  points  rise  and  the  alcohols  become  less  soluble  with 
increasing  molecular  weight.  The  specific  gravity,  however, 
does  not  decrease  as  in  the  case  of  homologous  fatty  acids. 

CHOLESTEROL  AND  RELATED  ALCOHOLS 

Cholesterol,  C26H44O,  is  the  characteristic  constituent  of  the 
unsaponifiable  matter  of  animal  fats.  It  is  only  sparingly 
soluble  in  cold  dilute  alcohol  or  cold  petroleum  ether,  but 


170 


METHODS  OF  ORGANIC  ANALYSIS 


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OILS,    FATS,    AND   WAXES 


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172  METHODS    OF   ORGANIC   ANALYSIS 

dissolves  readily  in  ether,  chloroform,  carbon  bisulphide,  fatty 
oils,  turpentine,  or  hot  strong  alcohol. 

Phytosterol  and  sitosterol  are  alcohols,  probably  isomeric  with 
cholesterol  and  resembling  it  closely  in  physical  properties, 
which  occur  in  vegetable  fats,  the  latter  being  found  especially 
in  the  oils  of  the  cereal  grains.  The  identification  of  one  of 
these  alcohols  may,  therefore,  aid  in  determining  the  origin  of  an 
oil  or  fat  or  in  the  detection  of  vegetable  fats  present  as  adul- 
terants in  the  more  expensive  fats  of  animal  origin,  as  will  be 
explained  in  the  next  chapter. 

REFERENCES 


ALDER- WRIGHT  and  MITCHELL  :  Animal  and  Vegetable  Fixed  Oils,  Fats, 

Butters,  and  Waxes. 

ALLEN  :  Commercial  Organic  Analysis,  Vol.  II. 
BENEDIKT  and  ULZER  :  Analyse  der  Fette  und  Wachsarten. 
GILL  :  Short  Handbook  of  Oil  Analysis. 
HEFTER  :  Technologic  der  Fette  und  Oele. 
HOPKINS  :  Oil-Chemists'  Handbook. 
LEACH  :  Food  Inspection  and  Analysis. 
LEWKOWITSCH  :  Chemical  Technology  and  Analysis  of  Oils,  Fats,  and  Waxes. 

Laboratory  Companion  to  Fat  and  Oil  Industries. 
LUNGE  :  Chemisch-technische  Untersuchungsmethoden. 
UBBELOHDE  :  Handbuch  der  Chemie  und  Technologic  der  Oele  und  Fette. 
WRIGHT  :  Analysis  of  Oils  and  Allied  Substances. 

II 

1902.  HUNT  :  A  Comparison  of  Methods  used  to  determine  Iodine  Values 

of  Oils.     J.  Soc.  Chem.  Ind.,  21,  454. 

TOLMAN  and  MUNSON:  Refractive  Indices  of  Salad  Oils.      /.  Am. 
Chem.  Soc.,  24,  754. 

1903.  LYTHGOE  :  Zeiss  Butyro-refracto meter  Readings  of  Edible  Oils  and 

Fats.     Technology  Quarterly,  16,  222. 

TOLMAN  and  MUNSON  :  Iodine  Absorption  of  Oils  and  Fats.     J.  Am. 
Chem.  Soc.,  25,  244. 

1905.  LYTHGOE  :  Refraction  Indices  of  Oils.     J.  Am.  Chem.  Soc.,  27,  887. 

1906.  DUNLAP  :  Preparation  of  Aldehyde-free  Ethyl  Alcohol  for  use  in  Oil 

and  Fat  Analysis.     J.  Am.  Chem.  Soc.,  28,  395. 

HALLER  :  Alcoholysis  of  Fatty  Substances.     Compt.  rend.,  143,  657, 
803. 


OILS,    FATS,    AND   WAXES  173 

RADCLIFF  :  Analytical  Constants  of  Carnaiiba  Wax.     J.  Soc.  Chem. 

Ind.,  25,  158. 
SCHNEIDER  and  BLUMENFELD  :   Characteristics   of  Certain  Animal 

Fats.     Chem.  Ztg.,  30,  53. 
THOMPSON  and  DUNLOP:  (Revision  of  Iodine  Numbers).     Analyst, 

31,  281. 

1907.  BERG  :  Examination  of  Beeswax.     Chem.  Ztg.,  31,  337. 

DONS  :  Index  of   Refraction   of   Fats  and   Fatty  Acids.     Z.   Nahr. 

Genussm.,  13,  257. 
Louis  and  SAUVAGE  :  New  Characteristic  Constant  of  Oils.     Compt. 

rend.,  145,  183. 
MEYER  ;  Determination  of  Unsaponifiable  Matter.     Chem.  Ztg.,  31, 

423. 

RAKUSIN  :  Optical  Properties  of  Animal  Fats.     Chem.  Ztg.,  30,  1247. 
RICHMOND  :  Temperature  Corrections  of  the  Zeiss  Butyro-refractom- 

eter.     Analyst,  32,  44. 
RICHTER  :  Maumene  Test  and  Iodine  Number  of  Certain  Oils.     Z. 

angew.  Chem.,  37,  1605. 
SCHICHT  and  HALPERN  :  Estimation   of    Unsaponifiable  Matters  in 

Fats.     Chem.  Ztg.,  31,  279. 
TWITCHELL  :  A  Reagent  in  the  Chemistry  of  Fats.     J.  Am.  Chem. 

Soc.,  29,  566. 

1908.  BERG  :  Analytical  Chemistry  of  Beeswax.     Chem.  Ztg.,  32,  777. 
FAHRION  :  Progress  in  Fat  Analysis  in  1907.     Z.  angew.  Chem.,  1908, 

1125. 
HALLA  :  Preparation  of  Alcoholic  Potassium  Hydroxide.     Chem.  Ztg. 

32,  890. 

INGLE  :  Notes  on  Wijs  Solution.     /.  Soc.  Chem.  Ind.,  27,  314. 

1909.  BARTLETT  and  SHERMAN  :  Effect  of  Excess  of  Reagent  and  Time  of 

Reaction  in  the  Determination,  of  Iodine  Numbers  of  Fatty  Oils. 
School  of  Mines  Quarterly,  31,  55. 

BOMER  :  Mixed  Glycerides  of  Palmitic  and  Stearic  Acids.  Z.  Nahr. 
Genussm.,  17,  353. 

BOYNTON  and  SHERMAN  :  A  Comparison  of  the  Calculated  and  De- 
termined Values  for  the  Specific  Temperature  Reactions  of  Oil 
Mixtures  with  Sulphuric  Acid.  School  of  Mines  Quarterly,  31,  64. 

FREUDLICH  :   Analytical  Methods  in  Stearin  Manufacture.      Chem. 

Rev.  Fette-  Harz.-Ind.,  15,  224,  246,  277;   Chem.  Abs.,  3,  253. 
1911.   COMMITTEE  REPORT:    (Methods  for  Moisture,  Volatile  Matter,  Sus- 
pended  Impurities,  Free    Fatty   Acids,    Unsaponifiable   Matter, 
Metallic  Soaps,  and  Titer  Test  in  Fats  and  Fatty  Oils).    J.  Lid. 
Eng.  Chem.,  3,  50. 

FAHRION:  Fat  Analysis  and  Fat  Chemistry  in  1910.  Z.  angew. 
Chem.,  24,  241. 


CHAPTER   IX 
Edible  Oils  and  Fats 

SALAD   OILS 

MOST  salad  oils  ai;e  sold  as  olive  oil.  The  principal  substitutes 
and  adulterants  are  cottonseed,  arachis  (peanut),  sesame,  maize, 
poppyseed,  and  lard  oils.  Both  quantitative  and  qualitative 
methods  must  be  used  in  any  thorough  examination  of  an  oil 
for  adulterants.  As  a  rule  an  oil  should  be  pronounced  adul- 
terated only  when  quantitative  determinations  yield  results  which 
could  not  be  obtained  from  a  pure  oil  of  normal  character. 
Qualitative  tests  are  usually  required  to  show  which  of  several 
possible  adulterants  is  present.  As  yet  certain  color  reactions 
are  indispensable  for  this  purpose,  but  in  using  these  tests  and 
interpreting  the  results,  it  must  be  remembered  that  they  de- 
pend upon  the  presence  of  constituents  which  may  be  removed 
or  destroyed  by  improved  methods  of  refining,  and  that  olive 
oil  which  has  been  altered  by  long  exposure  to  air  in  loosely 
stoppered  or  partially  filled  vessels,  or  oil  containing  a  small 
amount  of  some  accidental  impurity,  may  give  a  reaction  which 
cannot  be  distinguished  from  that  of  the  adulterant  sought. 
The  results  of  even  the  best  of  the  color  reactions  must  there- 
fore be  interpreted  with  great  caution  and  must  usually  be 
regarded  as  of  much  less  significance  than  the  quantitative 
numbers. 

On  the  other  hand,  a  good  grade  of  arachis  oil,  or  a  carefully 
prepared  mixture  of  lard  oil  with  one  of  the  seed  oils,  can  be 
added  to  olive  oil  in  large  proportion  without  affecting  the 
ordinary  constants  to  such  an  extent  as  to  pass  the  limits  which 
can  fairly  be  regarded  as  normal.  It  is  necessary,  therefore, 
not  only  to  compare  each  number  found  with  the  established 

174 


EDIBLE    OILS    AND    FATS  175 

limits  for  pure  oil,  but  also  to  view  the  quantitative  results  in 
their  relations  to  each  other  and  to  the  indications  of  the  quali- 
tative tests. 

While  the  system  to  be  followed  and  the  number  of  tests  re- 
quired will  naturally  vary  in  different  laboratories,  the  follow- 
ing may  be  recommended.  Determine  accurately  the  specific 
gravity  or  the  index  of  refraction,  the  iodine  number,  and  sa- 
ponification  number.  If  an  abundance  of  the  sample  is  at  hand, 
determine  the  specific  temperature  reaction  and  the  acidity. 
Apply  the  nitric  acid  test,  Halphen's  test,  one  or  more  of  the 
color  reactions  for  sesame  oil,  and  examine  for  arachidic  acid 
by  Renard's  method.  Finally,  if  the  importance  of  the  sample 
justifies  the  time  acquired,  separate  and  examine  the  unsaponi- 
fiable  matter  and  the  mixed  fatty  acids  and  determine  the  vis- 
cosity of  the  soap  solution  by  Abraham's  method  described 
below. 

ANALYTICAL  PROPERTIES  OF  OLIVE  OIL 

The  numbers  included  in  the  table  of  "constants"  already 
given  are  intended  to  cover  the  range  of  normal  variations  in 
oils  found  in  the  American  markets.  Miintz,  Durand,  and 
Milliau1  examined  samples  from  Africa,  Spain,  Portugal, 
Greece,  Turkey,  and  the  Levant  without  finding  any  significant 
variation  in  the  specific  gravity,  iodine  number,  or  temperature 
reaction.  According  to  Milliau,  Bertainchand,  and  Malet, 2 
however,  Tunis  oils  have  high  specific  gravities,  the  average  of 
49  samples  being  0.9183.  Tolman  and  Munson  have  recently 
examined  a  large  number  of  olive  oils  many  of  which  were  of 
known  origin.  The  following  are  taken  from  their  results:  3 

The  average  iodine  number  of  the  oils  from  California  is 
therefore  higher  than  that  of  the  French  and  Italian  oils  and, 
as  might  be  expected,  the  higher  iodine  number  is  accompanied 

1  Bulletin  du  Ministere  de  PAgriculture,  1895.     Quoted  from  Bui.  77,  Bur. 
Chem.,  U.  S.  Dept.  Agriculture. 

2  Bulletin  de  PAgriculture  et  Commerce  de  Tunis.     Quoted  from  Bull.  77, 
loc.  cit. 

3  Bui.  77,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 


176  METHODS   OF   ORGANIC   ANALYSIS 

TABLE  16.  —  ANALYTICAL  PROPERTIES  OF  OLIVE  OILS 


Description 

Iodine 
number 

gl»*«  n 

<**  c 
S.2 

8l| 

^•^s 

l-<  £ 

Specific 
temperature 
reaction 

Free  acid 

as  oleic 
per  cent 

California  oils  of  known  origin      1         ' 
(42  samples).                                  |Avg< 

89.8 
78.5 
85.3 

0.9180 
0.9162 
0.9170 

1.4718 
1.4703 
1.4713 

109.7 
94.5 
101.8 

8.211 
0.20 
1.20 

Italian    oils    of    known    origin 

Max. 
Min. 

86.1 
79.2 

0.9180 
0.9155 

1.4713 
1.4705 

104.7 
95.6 

2.79 
0.57 

(17  samples). 

Avg. 

81.6 

0.9163 

1.4709 

99.1 

1.11 

Italian  oils  (commercial)  not  found 
adulterated  (57  samples).2 

Max. 
Min. 

Avg. 

84.5 

77.5 
80.9 

0.9179 
0.9150 
0.9161 

1.4712 
1.4701 
1.4706 

108.4 
88.4 

97.8 

5.30 
0.72 

2.42 

French     oils     (commercial)     not 
found  adulterated  (60  samples). 

Max. 
Min. 

Avg. 

85.0 
79.0 
81.3 

0.91833 
0.9150 
0.9166 

1.4713 
1.4699 
1.4708 

114.4 
90.4 
100.1 

3.63 
0.45 
1.59 

by  a  higher  specific  gravity,  refractive  index,  and  temperature 
reaction.  Although  individual  samples  will  show  slight  varia- 
tions, the  same  general  relation  is  found  on  comparing  oils 
which  differ  in  iodine  numbers  though  obtained  from  the  same 
locality.  Thus  taking  at  random,  from  among  the  California 
oils  of  known  origin  examined  by  Tolman  and  Munson,  10  sam- 
ples with  high  and  10  with  low  iodine  numbers  the  following 
average  figures  were  found  : 


Iodine  number 

Specific  gravity 
15.5° 
15.5° 

Index  of 
refraction 
15.5° 

Specific 
temperature 
reaction 

First  group    .     .... 
Second  group     .    «    , 

88.44 
83.12 

0.9172 
0.9166 

1.4716 
1.4711 

105.7 

98.6 

1  Two  samples  with  larger  amounts  of  free  acids  were  found,  but  were  ex- 
cluded from  average  as  being  unfit  for  use  as  salad  oils. 

2  Omitting  one  sample  containing  15.25  per  cent  of  free  acid  and  having  a 
specific  gravity  of  0.9134. 

3  Omitting  one  sample  having  an  abnormally  high  specific  gravity  (0.9196) 
which  may  have  been  due  to  exposure. 


EDIBLE    OILS   AND    FATS  177 

The  normal  relations  of  the  constants  to  each  other  and  the 
changes  which  may  occur  as  the  result  of  age  or  exposure1 
must  always  be  taken  into  consideration  when  interpreting  the 
results  of  an  analysis.  For  records  of  individual  samples  of 
olive  oil  showing  iodine  numbers  of  90.5  to  94.7  see  Allen's  Com- 
mercial Organic  Analysis,  Fourth  Edition,  II,  113. 

The  standard  adopted  by  the  United  States  Department  of 
Agriculture  is  as  follows  : 

Olive  oil  is  the  oil  obtained  from  the  sound,  mature  fruit  of 
the  cultivated  olive  tree  (Olea  europcea  L.)  and  subjected  to 
the  usual  refining  processes ;  is  free  from  rancidity ;  has  a 
refractive  index  (25°  C.)  not  less  than  1.4660  and  not  exceed- 
ing 1.4680 ;  and  an  iodine  number  not  less  than  79  and  not 
exceeding  90. 

DETECTION  OF  COTTONSEED  OIL 

The  presence  of  cottonseed  oil  in  olive  oil  raises  the  specific 
gravity,  iodine  number,  and  temperature  reaction  and  lowers 
the  viscosity  of  the  soap  solution.  A  mixture  of  cottonseed 
and  lard  oils  may,  however,  be  added  to  olive  oil  in  large  quan- 
tity without  greatly  affecting  any  but  the  last  of  these  "  con- 
stants." The  qualitative  tests  for  cottonseed  oil  are  therefore 
of  considerable  importance. 

Halpherts  Reaction* 

Dissolve  1  part  of  sulphur  in  100  parts  of  carbon  bisulphide 
and  mix  the  solution  with  an  equal  volume  of  amyl  alcohol. 

Mix  equal  volumes,  2  to  3  cc.  each,  of  the  reagent  and  the 
oil  to  be  tested  and  heat  the  test  tube  containing  the  mixture 
gently  at  first  until  violent  boiling  ceases,  then  in  a  bath  of 
boiling  saturated  solution  of  common  salt.  Heat  for  2  hours 
unless  a  color  develops  sooner.  If  cottonseed  oil  is  present,  the 
solution  turns  orange  or  red. 

This  is  probably  the  most  sensitive  and  characteristic  test  for 

1  See  section  on  this  subject  beyond. 

2  Halphen  :  Ann.  chim.  anal.,  1898,  3,  9  ;  Analyst.,  1898,  23,  131 ;  Bui.  107. 
Bur.  Chem.,  U.  S.  Dept.  Agriculture. 

N 


178  METHODS   OF   ORGANIC    ANALYSIS 

cottonseed  oil  and  the  least  liable  to  give  unsatisfactory  results 
in  the  hands  of  an  inexperienced  person.  The  presence  of  1  or 
2  per  cent  of  unchanged  cottonseed  oil  in  olive  oil  is  detected 
without  difficulty.  A  reaction  is  often  obtained l  with  lard  or 
lard  oil  or  even  with  butter  fat2  from  animals  which  have  been 
fed  upon  cottonseed  meal.  Copac  (or  kapok)  oil,  which  is 
closely  related  to  cottonseed  oil,  gives  the  same  reaction.  When 
heated  at  250°  for  10  to  20  minutes,  cottonseed  oil  loses  the  prop- 
erty of  giving  this  reaction.  No  pure  olive  oil  has  yet  been  known 
to  give  a  similar  coloration.  Hence  a  positive  result  is  con- 
sidered conclusive,  but  a  negative  result  is  not.  The  substance 
to  which  the  reaction  is  due  cannot  be  removed  by  treatment 
with  animal  charcoal3  and  is  supposed  to  be  an  unsaturated 
acid  which  combines  with  sulphur  giving  a  red  compound.  4 

Nitric  Acid  Test 

Cottonseed  oil  shaken  at  room  temperature  with  an  equal 
volume  of  nitric  acid,  of  1.37  to  1.38  specific  gravity,  gives 
a  brown  coloration,  sometimes  only  on  standing  overnight. 
Other  seed  oils  give  similar  reactions.  Normal  olive  oil  under 
the  same  treatment  shows  no  change  of  color. 

The  test  is  not  so  delicate  as  that  of  Halphen,  but  is  applicable 
to  cottonseed  oil  which  has  been  heated  until  it  no  longer  colors 
the  Halphen  reagent.  *  It  may  also  be  of  value  in  determining 
whether  a  weak  test  with  the  latter  reagent  is  due  to  a  small 
amount  of  unheated  cottonseed  oil  or  to  a  larger  amount  which 
has  been  heated  sufficiently  to  weaken  the  Halphen  reaction 
(Tolman  and  Munson).  According  to  Lewkowitsch  this  re- 
action cannot  be  relied  upon  to  detect  less  than  10  to  20  per 
cent  of  American  cottonseed  oil  in  olive  oil.  Tolman  and 
Munson  consider  the  test  much  more  delicate.  A  positive  re- 

iSoltsein:  Z.  offentl  Chem.,  1901,  7,  140. 

2Wauters:  Bull.  Assoc.  Belg.  Chem.,  13,  404;  J.  Soc.  Chem.  Ind.,  1900, 
19,  172. 

8Utz  :  Chem.  Rev.  Fett.-Harz-Ind.,  1902,  9,  125  ;  Gill's  Oil  Analysis,  p.  73. 

*  Raikow  :  Chem.  Ztg.,  1900,  24,  562,  583  ;  1902,  26,  10.  See  also  Halphen  : 
Bull.  Soc.  Chim.,  1905,  [3],  33,  108. 


EDIBLE    OILS   AND    FATS  179 

suit  should  always  be  confirmed  by  finding  a  high  iodine 
number  or  by  proving  the  presence  of  some  other  oil  having  a 
very  low  iodine  number ;  for  pure  olive  oil  if  much  altered  as 
the  result  of  age  and  exposure  will  sometimes  give  a  reaction 
which  cannot  be  distinguished  from  that  of  cottonseed  oil. 

DETECTION  OF  ARACHIS  (PEANUT)  OIL 

Arachis  oil  has  usually  a  higher  specific  gravity  and  iodine 
number  and  practically  always  a  higher  temperature  reaction 
than  olive  oil.  The  specific  temperature  reaction  of  arachis  oil 
with  concentrated  sulphuric  acid  is  usually  40  to  60  units 
higher  than  the  iodine  number  of  the  same  sample,  whereas 
olive  oil  usually  shows  a  difference  of  less  than  20  and  very 
rarely  of  more  than  25  units  between  the  iodine  and  the  specific 
Maumene  numbers.  The  presence  of  peanut  oil  in  olive  oil 
greatly  diminishes  the  viscosity  of  the  soap  solution  obtained 
on  saponification.  Any  of  these  changes,  however,  might  be 
due  to  other  adulterants.  Arachis  oil  is  shown  conclusively  by 
isolating  and  identifying  arachidic  acid.  This  can  be  done 
with  approximately  quantitative  results  by  Tolman's  modifica- 
tion of  Renard's  method. 

Renard-Tolman  Test  for  Arachidic  Acid1 

Weigh  20  grams  of  oil  in  an  Erlenmeyer  flask.  Saponify 
with  alcoholic  potash,  neutralize  exactly  with  dilute  acetic  acid, 
using  phenolphthalein  as  indicator,  and  wash  the  solution  into 
a  500-cc.  flask  containing  a  boiling  mixture  of  100  cc.  of  water 
and  120  cc.  of  a  20  per  cent  lead  acetate  solution.  Boil  one 
minute  and  then  cool  by  immersing  the  flask  in  water,  occa- 
sionally giving  it  a  whirling  motion  to  cause  the  precipitated 
lead  soaps  to  stick  to  the  sides  of  the  flask.  After  thorough 
cooling,  pour  off  the  water  containing  the  excess  of  lead  acetate 
and  wash  the  soap  with  cold  water  and  then  with  90  per  cent 
alcohol.  After  pouring  off  the  alcohol  as  completely  as 

1  Renard  :  Compt.  rend.,  1871,  73,  1330. 

Tolman  :  U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  81,  p.  64.  See  also 
Archbutt:  J.  Soc.  Chem.  2nd.,  1898,  17,  1124. 


180  METHODS   OF   ORGANIC    ANALYSIS 

possible,  add  200  cc.  of  ether,  cork  the  flask  and  allow  to  stand 
until  the  soap  is  disintegrated,  then  connect  with  a  reflux  con- 
denser, heat  gently  to  boiling,  and  boil  for  5  minutes  on  a 
safety  water  bath  or  an  electric  heater.  Cool  to  15°  and  allow 
to  stand  overnight. 

Filter,  wash  the  residue  thoroughly  with  ether,  and  then 
transfer  it  from  the  filter  to  the  flask  by  means  of  a  stream  of 
hot  water  acidified  with  hydrochloric  acid.  Add  an  excess  of 
dilute  hydrochloric  acid  and  200  cc.  of  hot  water  and  heat  until 
the  fatty  acids  separate  as  a  clear  oily  layer.  Nearly  fill  the 
flask  with  hot  water  and  allow  to  stand  at  room  temperature 
until  the  layer  of  fatty  acids  has  completely  separated  and  solid- 
ified. Remove  and  drain  the  cake  of  fatty  acids,  wash  again 
with  hot  water,  then  dissolve  in  100  cc.  of  boiling  alcohol,  90 
per  cent  by  volume.  Cool  the  solution  to  15°,  shaking  fre- 
quently, and  allow  it  to  stand  as  long  as  any  acid  continues  to 
crystallize  out,  or  overnight,  at  a  temperature  not  above  20°. 
Filter,  wash  the  crystals  twice  with  10  cc.  of  90  per  cent 
alcohol,  noting  the  total  volume  of  filtrate  and  washings,  and 
then  with  alcohol,  70  per  cent  by  volume  (in  which  the  crystals 
are  practically  insoluble).  Dissolve  the  crystals  by  means  of 
hot  absolute  alcohol  in  a  weighed  dish,  evaporate,  dry,  and 
weigh.  To  this  weight  add  0.0045  gram  for  each  10  cc.  of  90 
per  cent  alcohol  in  the  filtrate  and  washings  if  the  temperature 
of  filtration  was  20°;  or  0.0025  gram  for  each  10  cc.  if  the  tem- 
perature was  15°. 

The  melting  point  of  arachidic  acid  obtained  in  this  way  is 
71°  to  73°.  According  to  Tolman  and  Munson 1  the  determina- 
tion of  the  melting  point  must  not  be  neglected  since  cotton- 
seed and  lard  oils  have  been  found  to  give  crystals  resembling 
arachidic  acid  in  appearance,  but  having  a  lower  melting  point. 
Tolman  finds  that  from  5  to  10  per  cent  of  the  oil  can  be  de- 
tected by  this  method.  On  the  usual  assumption  that  the  oil 
yields  5  per  cent  of  the  acid,2  each  centigram  found  as  described 

1  U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  77,  p.  35. 

2  This  is  probably  more  nearly  a  maximum  than  an  average  yield.     Tolman 
and  Munson  (loc.  cit.)  obtained  from  3.41  to  4.24  per  cent. 


EDIBLE    OILS   AND    FATS  181 

above  (using  20  grams  of  oil)  indicates  1  per  cent  of  arachis 
(peanut)  oil  in  the  sample. 

Archbutt  (Allen's  Commercial  Organic  Analysis,  4th  Ed.,  II, 
99-100)  recommends  the  following  method  of  Bellier  1  as  giving 
satisfactory  qualitative  results. 

Solutions.  —  (1)  Alcoholic  potassium  hydroxide,  made  by 
dissolving  8.5  grams  pure  potassium  hydroxide  in  70  per  cent 
alcohol  and  making  up  to  100  cc. 

(2)  Acetic  acid  of  such  strength  that  1.5  cc.  will  exactly 
neutralize  5  cc.  of  the  potash  solution  (about  28-29  per  cent  of 
actual  acetic  acid). 

Test.  —  Weigh  1  gram  of  sample  into  a  dry  test  tube,  add 
5  cc.  of  the  potash  solution,  and  boil  gently,  avoiding  evapora- 
tion, over  a  free  flame  until  saponification  is  complete,  probably 
a  little  over  2  minutes;  then  add  1.5  cc.  acetic  acid,  or  just 
sufficient  to  neutralize  the  alkali,  mix  well,  cool  rapidly  in 
water  at  17°  to  19°,  and  let  stand  at  this  temperature  for  at 
least  30  minutes,  shaking  occasionally ;  then  add  50  cc.  70  per 
cent  alcohol  containing  1  per  cent  by  volume  of  hydrochloric 
acid  of  1.16  sp.  gr.,  shake  well,  and  again  place  in  the  cold 
water  for  1  hour.  In  the  absence  of  arachis  oil  the  liquid  should 
remain  clear  or  become  merely  opalescent,  while  if  the  sample 
contained  more  than  10  per  cent  of  arachis  oil  a  flocculent, 
crystalline  precipitate  is  obtained. 

DETECTION  OF  SESAME  OIL 

Sesame  oil  affects  the  usually  determined  constants  in  the 
same  way  as  cottonseed  oil  and  to  practically  the  same  extent. 
The  color  reactions  are  usually  considered  quite  characteristic. 

Baudouin's   Test 

Dissolve  0.1  gram  of  sugar  in  10  cc.  of  hydrochloric  acid  of 
1.18  to  1.20  specific  gravity  and  add  20  cc.  of  the  oil.  Shake 
thoroughly  in  a  test  tube  for  one  minute  and  allow  to  stand. 
The  water  solution  separates  quickly  and  shows  a  distinct  red 

1  Ann.  chim.  anal.,  1899,  4,  4. 


182  METHODS   OF   ORGANIC    ANALYSIS 

or  "crimson  color  if  the  sample  contains  1  per  cent  or  more  of 
sesame  oil.  The  active  reagent  is  probably  furfural  formed  by 
the  action  of  the  acid  upon  the  sugar. 

Villivecchia's  modification  consists  in  shaking  10  cc.  of  the 
oil  with  10  cc.  of  hydrochloric  acid  (1.20  sp.  gr.)  to  which  has 
been  added  0.1  cc.  of  a  2  per  cent  solution  of  furfural  in  95  per- 
cent alcohol. 

Olive  oils  of  known  purity  have  usually  been  found  to  give 
only  a  slight  pink  color,  but  sometimes  the  reddening  of  the 
water  solution  is  so  pronounced  as  to  cause  confusion  with  that 
produced  by  a  small  amount  of  sesame  oil.  Check  experiments 
therefore  should  always  be  made.  If  much  sesame  oil  is  present, 
the  red  color  should  be  perceptible  in  the  oily  layer  as  well  as 
in  the  water  solution. 

Tochers  Test 

Dissolve  1  gram  of  pyrogallol  in  15  cc.  of  concentrated  hydro- 
chloric acid.  Shake  this  solution  with  15  cc.  of  oil  in  a  sep- 
aratory  funnel  and  allow  to  stand  for  1  or  2  minutes.  Draw 
off  the  aqueous  solution  and  boil  for  5  minutes.  The  presence 
of  sesame  oil  is  indicated  if  the  solution  after  boiling  appears 
red  by  transmitted  and  blue  by  reflected  light. 

This  test  has  not  been  so  generally  used  nor  so  thoroughly 
studied  as  the  preceding.  The  Association  of  Official  Agricul- 
tural Chemists  authorize  the  use  of  either  Baudouin's,  Villivec- 
chia's,  or  Tocher's  test  for  the  detection  of  sesame  oil  in  edible 
oils  and  fats. 

DETECTION  OF  MAIZE,  POPPYSEED,  AND  LARD  OILS 

Each  of  these  oils  has  a  characteristic  odor  or  taste,  the  odor 
of  lard  oil  being  intensified  by  heating.  These  properties, 
however,  cannot  be  relied  upon,  as  the  substances  to  which  they 
are  due  can  be  almost  entirely  eliminated  in  the  refining 
process.  The  effect  of  maize  or  poppy  oil  in  raising  the  iodine 
number  would  be  very  noticeable,  but  might  be  neutralized  by 
the  addition  of  a  somewhat  greater  quantity  of  lard  oil.  The 
difference  between  the  iodine  number  and  the  specific  tern- 


EDIBLE   OILS   AND   FATS  183 

perature  reaction  would  be  appreciably  greater  in  such  a  mix- 
ture than  in  pure  olive  oil  (compare  detection  of  arachis  oil). 
Maize  and  poppyseed  oils  react  with  nitric  acid,  giving  brown 
colors  similar  to  that  produced  by  cottonseed  oil.  Lard  oil 
often  gives  the  same  reaction  and  might  be  indicated  by  the 
melting  point  of  the  fatty  acids  (those  of  lard  oil  having  a 
relatively  high  melting  point,  33°  to  38°  according  to  Tolman 
and  Munson)  or  by  the  character  of  the  unsaponifiable  matter 
—  phytosteryl  acetate  test.1  (See  references  at  the  end  of  this 
chapter.) 

All  of  these  oils  (as  well  as  most  others)  yield  soap  solutions 
of  much  lower  viscosity  than  those  obtained  from  pure  olive 
oils.2  The  determination  and  significance  of  this  property 
has  been  studied  in  some  detail 3  and  the  method  has  been 
found  capable  of  giving  valuable  results,  especially  if  the  con- 
ditions worked  out  by  Abraham  are  carefully  observed.  For 
a  full  discussion  of  these  conditions  the  original  paper  must 
be  consulted.  The  essential  features  of  the  process  are  as 
follows : 

Abraham 's  Modification  of  Blasdales   Viscosity  Test 

The  saponification  number  having  been  determined,  weigh  3 
grams  of  oil  in  an  accurately  graduated  100-cc.  flask,  add  2  cc. 
of  alcohol  and  an  amount  of  standard  potash  solution  sufficient 
to  saponify  the  oil  and  leave  an  excess  of  0.625  gram  of  potas- 
sium hydroxide.  Close  the  flask  with  a  stopper  having  a 
Kroonig  valve  and  saponify  on  a  water  bath.  After  saponifi- 
cation expel  the  alcohol  by  warming  and  allowing  air  freed 
from  carbon  dioxide  to  pass  through  the  flask,  while  a  partial 
vacuum  is  maintained  by  means  of  a  suction  pump.  In  this 
way  the  alcohol  is  entirely  removed  in  5  to  10  minutes.  Evap- 
oration should  not  be  carried  to  complete  dryness.  Without 
allowing  the  flask  to  cool,  add  50  cc.  of  hot  water,  rotate  gently 

1  Bonier  :  Z.  Nahr.-Genussm.,  1901,  4,  1091. 

2  Blasdale  :  J.  Am.  Chem.  Soc.,  1895,  17,  937. 

3  Abraham  :  Ibid.,  1903,  25,  968.     Sherman  and  Abraham  :  Ibid.,  1903.  25, 

977. 


184  METHODS   OF   ORGANIC   ANALYSIS 

until  a  homogeneous  solution  of  the  soap  is  obtained,  cool  to 
20°,  fill  to  the  mark  with  distilled  water,  and  mix  well  by  shak- 
ing or  by  repeatedly  pouring  the  solution  from  one  flask  to 
another.  Determine  the  viscosity  of  the  solution  in  an  Ostwald 
viscosimeter  immersed  in  water  kept  at  20°.  Care  must  be 
taken  to  avoid  the  introduction  of  air  bubbles  into  the  vis- 
cosimeter and  to  maintain  the  exact  temperature.  Repeat  the 
readings  until  five  or  more  concordant  results  are  obtained.  The 
viscosimeter  is  standardized  by  means  of  distilled  water,  and 
it  is  advisable  to  select  for  this  work  an  instrument  in  which 
the  time  of  flow  of  water  is  about  100  seconds.  Successive 
readings  of  a  soap  solution  should  then  agree  within  2  seconds. 
The  viscosity  is  calculated  as  follows : 

vl  =  1001 

where 

v1  =  the  viscosity  number. 
£j  =  the  time  of  flow  (in  seconds). 
s1  —  specific  gravity  of  the  solution. 
t  =  time  of  flow  of  distilled  water. 

The  viscosity  numbers  obtained  by  this  method  were : 

Olive  oil  of  known  purity  (9  samples) 168.0-185.7 

Olive  oil  of  doubtful  purity  (4  samples) 145.8-165.8 

Lard  oil  (5  samples) 122.9-135.0 

Arachis  (peanut)  oil  (1  sample) 126.6 

Cottonseed  oil  (3  samples) 126.6-127.9 

Rapeseed  oil  (3  samples) 124.7-125.7 

Sesame  oil  (1  sample) 139.2 

Maize  oil  (1  sample) 126.6 

Poppyseed  oil  (1  sample) 123.9 

Mixtures  of  olive  and  lard  oils  gave  viscosity  numbers  agree- 
ing closely  with  those  obtained  by  calculation,  but  cottonseed 
or  arachis  oil  when  added  to  olive  oil  lowered  the  viscosity 
to  a  much  greater  extent  than  would  have  been  predicted, 
indicating  that  the  viscosity  number  is  a  more  useful  means 
of  detecting  adulteration  than  appears  from  a  comparison  of 


EDIBLE    OILS    AND    FATS  185 

the  results  obtained  on  testing  the  olive  oil  and  its  adulterants 
separately. 

The  usefulness  of  the  method  for  testing  isolated  samples  is 
limited  by  the  fact  that  comparable  results  can  be  obtained 
only  under  strictly  uniform  conditions,  the  viscosity  of  the  soap 
solution  being  greatly  influenced  by  slight  variations  in  strength, 
alkalinity,  or  temperature,  while  the  terms  in  which  the  results 
are  expressed  will  naturally  vary  with  the  form  of  viscosimeter 
used ;  but  in  cases  of  sufficient  importance  to  justify  the  time 
required  to  arrange  the  apparatus  and  make  comparative  deter- 
minations, the  "  viscosity  number  "  will  be  found  an  important 
factor  in  the  examination  of  olive  oil  for  adulterants. 

BUTTER 

Butter  is  officially  defined1  as  "the  product  made  by  gathering 
in  any  manner  the  fat  of  fresh  or  ripened  cream  into  a  mass  which 
also  contains  a  small  portion  of  the  other  milk  constituents,  with 
or  without  salt."  Standard  butter  contains  not  less  than  82.5 
per  cent  of  butter  fat,  having  a  Reichert-Meissl  number  not  less 
than  24  and  a  specific  gravity  not  less  than  0.905  at  40°/40°. 
By  acts  of  Congress  approved  August  2, 1886,  and  May  9,  1902, 
butter  may  also  contain  additional  coloring  matter. 

Butter  may  fail  to  meet  the  requirements  of  the  official  stand- 
ard either  because  of  a  deficiency  in  the  percentage  of  fat  or  be- 
cause of  the  presence  of  foreign  fat  or  butter  fat  of  abnormal 
character,  though  the  sample  may  contain  nothing  which  can  be 
regarded  as  unwholesome.  Butter  analysis  therefore  includes 
(1)  the  examination  of  the  whole  butter,  (2)  the  examination 
of  the  butter  fat. 

The  methods  of  examining  butter  fat  will  be  given  fully  be- 
low, but  for  convenience  the  determination  of  water,  fat,  curd, 
and  ash  will  be  described  first.  The  point  at  which  tests  for 
preservatives  can  conveniently  be  made  will  be  indicated,  but 
the  detection  of  foreign  colors  will  not  be  considered,  as  these 
are  not  regarded  as  adulterants. 

1  Circular  No  19,  Office  of  the  Secretary,  U.  S.  Dept.  Agriculture. 


186  METHODS  OF  ORGANIC  ANALYSIS 

DETERMINATION  OF  WATER,  FAT,  CURD,  AND  ASH 

Especial  care  must  be  taken  in  sampling  butter,  since  the 
water,  salt,  and  curd  are  often  unevenly  distributed  and  an 
attempt  to  mix  by  stirring  is  apt  to  result  in  squeezing  out 
drops  of  brine.  If  a  large  quantity  is  to  be  sampled,  a  butter 
trier  should  be  used,  and  the  portions  thus  drawn  united  until 
a  working  sample  of  about  500  grams  is  obtained. 

Melt  the  sample  at  the  lowest  possible  temperature  in  a  wide- 
mouthed  glass-stoppered  bottle,  shake  violently  to  insure  a  homo- 
geneous mixture,  and  continue,  the  shaking  while  cooling  the  sample 
until  it  is  thoroughly  solidified.  Great  care  is  necessary  here  to 
prevent  a  separation  of  water  and  fat. 

Thoroughly  clean  and  dry  a  lipped  dish  or  beaker  having  a 
flat  bottom  of  at  least  20  square  centimeters.  Weigh  the  dish, 
introduce  1.5  to  2  grams  of  butter,  and  re  weigh  quickly  to  avoid 
evaporation.  Dry  to  constant  weight  in  a  boiling  water  oven. 
The  loss  is  water.  Treat  the  dry  residue  with  petroleum  ether 
or  benzine,  transfer  it  to  a  weighed  Gooch  crucible  having  a  felt 
of  ignited  asbestos,  and  wash  with  the  solvent  until  all  fat  is  re- 
moved. Dry  the  crucible  and  residue  to  constant  weight  in  a 
water  oven  or  an  air  bath  not  above  110°.  The  material  dis- 
solved by  the  petroleum  ether  or  benzine  is  fat.  Burn  the  curd 
at  a  temperature  below  a  red  heat  and  weigh  the  crucible  con- 
taining the  ash. 

Notes.  —  The  method  given  is  essentially  that  of  the  Associ- 
ation of  Official  Agricultural  Chemists.1  If  preferred  the  but- 
ter can  be  dried  on  clean  dry  sand  or  asbestos.  When  the  latter 
is  not  used,  it  is  important  that  the  butter  form  only  a  very  thin 
layer  on  the  bottom  of  the  dish  or  beaker  ;  otherwise  the  water 
sinks  into  the  melted  fat  and  is  only  very  slowly  expelled  at  the 
temperature  of  the  boiling  water  oven.  For  convenience  in 
transferring  and  washing  the  residue  with  petroleum  ether  the 
latter  should  be  used  in  a  small  wash  bottle  having  a  ground 
glass  stopper.  Instead  of  drying  in  a  dish  and  transferring  the 
residue  to  a  crucible,  the  butter  may.  be  weighed  directly  in  a 

1  Bui.  107,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 


EDIBLE    OILS   AND   FATS  187 

crucible  two  thirds  filled  with  fibrous  asbestos,  dried  to  constant 
weight,  and  then  extracted.1  A  device  for  facilitating  this  drying 
by  passing  a  current  of  air  through  the  heated  crucible  has  been 
described  by  Bird.2  References  to  a  number  of  other  quick 
methods  for  moisture  in  butter  will  be  found  at  the  end  of  the 
chapter. 

Great  care  must  be  taken  to  burn  the  curd  at  the  lowest  tem- 
perature possible  in  order  to  avoid  loss  of  chlorine  (see  Chapter 
XVII).  Any  small  amount  of  milk  sugar  which  the  butter 
might  contain  would  be  counted  as  curd  in  this  analysis.  The 
percentage  of  salt  can  be  found  by  determining  chlorine  in  the 
ash,  or  by  repeatedly  washing  the  butter  with  hot  water  in  a 
separatory  funnel  and  titrating  the  combined  washings  with  a 
standard  solution  of  silver  nitrate.  Good  butter  usually  con- 
tains 10  to  14  per  cent  water,  84  to  87  per  cent  fat,  0.5  to  1.5 
per  cent  curd,  2.0  to  4.0  per  cent  ash  if  salted  ;  if  unsalted, 
0.25  to  0.5  per  cent.  The  water  content  of  butter  has  often 
been  limited  by  legal  or  trade  standards  to  16  per  cent.  This  is 
considerably  above  the  present  average  for  creamery  butter,  as 
shown  by  an  investigation  made  by  the  United  States  Depart- 
ment of  Agriculture  in  1902. 3  Of  800  samples  from  400  cream- 
eries in  18  states,  the  average  water  content  was  11.78  per  cent; 
85  per  cent  of  the  samples  contained  between  10  and  14  per  cent; 
the  extreme  limits  were  7.20  and  17.62  per  cent.  The  amount  of 
salt  added  to  butter  varies  greatly  with  the  demands  of  differ- 
ent markets,  but  over  5  per  cent  would  be  excessive  unless  the 
butter  were  intended  for  export  to  a  tropical  country.  An  ex- 
cessive amount  of  curd  indicates  careless  manufacture  or  fraud- 
ulent increase  of  weight  and  is  likely  to  injure  the  keeping 
qualities  of  the  butter. 

1  Richards  and  Woodman:  Air,  Water,  and  Food  (2d  Ed.),  201. 

2  J.  Am.  Chem.  Soc.,  1905,  27,  818. 

3  Circular  No.  39,  Bureau  of  Animal  Industry  ;  Analyst,  1903,  28,  184. 


188  METHODS   OF   ORGANIC   ANALYSIS 

.  EXAMINATION  OF  BUTTER  FAT 

Preparation 

Melt  100  grams  or  more  of  the  butter  and  allow  it  to  stand 
at  45°  to  55°  until  the  water  and  salt  settle  to  the  bottom.1 
Pour  off  the  melted  fat  by  decantation  and  filter  it  through  a 
dry  paper  in  a  funnel  heated  by  a  water  jacket  or  supported  in 
a  drying  oven  kept  at  about  60°.  The  filtered  fat,  which  must 
be  free  from  turbidity,  is  received  in  a  wide-mouthed  bottle  and 
kept  stoppered  in  a  cool  place  until  analyzed. 

Reichert-Meissl  or  Reichert-Wollny  Number* 

This  number  is  a  comparative  measure  of  the  proportion  of 
volatile  acids,  and  is  the  most  important  basis  for  deciding  the 
purity  of  butter  fat.  Often  the  presence  or  absence  of  foreign 
fat  in  butter,  or  the  proportion  of  butter  fat  in  oleomargarine, 
is  inferred  from  this  number  alone.  In  the  interest  of  uni- 
formity of  results  official  chemists  have  sought  to  describe 
the  method  in  such  detail  as  to  eliminate  variations  due  to 
manipulation. 

Reagents.  —  1.  Caustic  soda  solution,  made  by  dissolving 
sodium  hydroxide  (nearly  free  from  carbonate)  in  an  equal 
weight  of  distilled  water. 

2.  Alcohol,   92  to   95  per  cent,   containing  no   appreciable 
amount  of  volatile  acid,  either  free  or  combined. 

3.  Dilute  sulphuric  acid,  made  by  mixing  pure  concentrated 
sulphuric  acid  with  four  times  its  volume  of  water. 

4.  An  accurately  standardized  approximately  tenth-normal 
solution  of  barium  (or  sodium)  hydroxide. 

5.  A  1  per  cent  solution  of  phenolphthalein  in  alcohol. 
Determination.  —  Thoroughly  clean  and  dry  a  flask  of  250  to 

1  This  water  solution  can  be  tested  for  preservatives,  of  which  boric  acid  and 
borax  are  most  likely  to  be  found  in  butter.  For  methods  see  Chapter  XVIII. 

2Reichert:  Z.  anal.  Chem.,  1879,  18,  69.  Meissl :  Dingier^  s  poly  tech. 
Journ.,  1879,  233,  229.  Wollny  :  Milch  Ztg.,  1887,  16,  609 ;  Analyst,  1887, 
12,  203,  235  ;  1888,  13,  8,  38.  These  papers  are  reprinted  in  Ephraim's  Origi- 
nal Arbeiten  iiber  Analyse  der  Nahrungsinittel.  . 


EDIBLE    OILS   AND    FATS  189 

800  cc.  capacity  Weigh  the  flask,  thoroughly  mix  the  melted 
fat,  introduce  5.6  to  5.8  cc.  measured  at  about  50°,  allow  the 
flask  and  fat  to  cool  for  15  to  20  minutes,  and  reweigh  (or,  if 
convenient,  weigh  exactly  5  grams  of  fat  into  the  flask).  Add 
10  cc.  of  the  alcohol  and  2  cc.  of  the  caustic  soda  solution,  attach 
the  flask  to  a  reflux  condenser,  and  boil  on  a  water  bath  or 
electric  heater  for  at  least  half  an  hour  to  insure  complete 
saponification.1 

Evaporate  the  alcohol  by  heating  the  flask  in  a  steam  bath, 
shaking  occasionally  to  avoid  danger  of  loss  from  frothing  and 
to  facilitate  the  removal  of  the  alcohol.  Add  132  cc.  of  recently 
boiled  distilled  water,  warm  at  60°  to  70°  until  the  soap  is  com- 
pletely dissolved,  add  8  cc.  of  the  dilute  sulphuric  acid  and  a 
few  pieces  of  pumice  stone,  re-stopper  the  flask  or  connect  it 
with  a  condenser,  and  warm  without  boiling  until  the  fatty  acids 
separate  as  a  clear  layer.  Distill 2  through  a  glass  condenser, 
collecting  the  distillate  in  a  flask  accurately  graduated  at  110  cc. 
The  distillation  should  be  so  regulated  that  110  cc.  will  be  col- 
lected in  from  28  to  32  minutes.  Mix  the  distillate,  filter 
through  dry  paper,  and  titrate  100  cc.  of  the  filtrate,  using  0.5 
cc.  of  the  phenolphthalein  solution  as  indicator,  until  the  red 
color  remains  apparently  unchanged  for  2  minutes.  Increase 
the  burette  reading  by  one  tenth  (on  account  of  the  10  cc.  of 
distillate  not  titrated),  and  calculate  the  number  of  cubic  centi- 
meters of  tenth-normal  alkali  which  would  have  been  required 
if  exactly  5  grams  of  fat  were  taken  for  the  determination. 
This  is  the  Reichert-Meissl  number. 

Notes.  —  The  number  thus  found  does  not  represent  the  total 
volatile  acids  present.  The  yield  is  fairly  uniform  if  the  given 
conditions  of  dilution  and  distillation  are  maintained.  Wollny 
submitted  this  method  to  an  exhaustive  examination  and 
pointed  out  the  following  sources  of  error  :  (1)  absorption  of 

1  The  saponification  can  also  be  accomplished  by  heating  in  a  closed  flask, 
using  either  aqueous  or  alcoholic  alkali,  or  by  means  of  the  glycerol-soda  solution 
proposed  by  Leffmann  and  Beam  :  Analyst,  1891,  16,  153. 

2  In  ordinary  work  a  form  of  apparatus  similar  to  that  shown  in  Fig.  1, 
Chapter  I,  may  be  used. 


190  METHODS   OF   ORGANIC   ANALYSIS 

carbon  dioxide  during  saponification,  (2)  formation  of  esters 
during  saponification,  (3)  formation  of  esters  during  distillation, 
(4)  coherence  of  fatty  acids  during  distillation,  resulting  in 
holding  back  some  of  the  volatile  acid,  (5)  variations  in  the 
proportion  of  volatile  acid  carried  over,  due  to  differences  in 
size  and  shape  of  distillation  apparatus.  In  order  to  avoid  dis- 
crepancies from  these  and  other  causes  he  published  an  elabo- 
rately detailed  system  of  manipulation  and  precautions.  It  has 
been  shown  that  Wollny  greatly  overestimated  the  probable  er- 
rors of  the  method  as  previously  carried  out  and  that  some  of 
his  precautions  are  unnecessary;  but  as  in  the  main  they  tend 
toward  greater  uniformity  of  results,  they  have  been  adopted 
with  slight  modifications  by  the  Association  of  Official  Agricul- 
tural Chemists,  whose  methods l  are  usually  accepted  as  stand- 
ard in  the  United  States  and  should  be  followed  exactly  in  any 
determination  which  is  likely  to  be  made  the  basis  of  legal 
action. 

In  Great  Britain,  a  joint  committee  representing  the  Govern- 
ment Laboratory  and  the  Society  of  Public  Analysts  has  adopted 
the  method  essentially  as  described  above  with  the  following 
specifications  for  the  apparatus  to  be  employed : 2  Flask  used  for 
saponification  and  distillation:  capacity,  300  cc.  ;  length  of 
neck,  7  to  8cm.;  width  of  neck,  2  cm.  The  flask  is  connected 
with  the  condenser  by  means  of  a  bent  glass  tube  7  mm.  wide,  so 
placed  that  the  bend  is  15  cm.  above  the  top  of  the  cork.  At 
a  distance  of  5  cm.  above  the  cork  is  a  bulb  5  cm.  in  diameter. 
The  flask  is  supported  on  a  circular  piece  of  asbestos  12  cm.  in 
diameter,  having  a  hole  5  cm.  in  diameter  in  the  center,  so  that 
the  bottom  of  the  flask  is  heated  by  a  free  flame  during  the  dis- 
tillation. The  British  committee  further  prescribed  that  blank 
determinations  be  made  and  the  volume  of  alkali  found 
necessary  to  neutralize  the  distillate  (which  volume  should  not 
exceed  0.3  cc.)  be  deducted  in  calculating  the  results  of  each 
determination.  The  number  so  obtained  is  called  the  Reichert- 
Wollny  number. 

1  Bui.  107,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 

2  Analyst,  1900,  25,  309. 


EDIBLE    OILS    AND    FATS  191 

The  Reichert-Meissl  or  Reichert-Wollny  number  of  butter 
fat  is  usually  between  24  and  34  ;  that  of  coconut  fat,  between 
6  and  8  ;  of  other  edible  fats  and  oils,  usually  less  than  1. 

Specific  Gravity 

The  specific  gravity  of  butter  fat  has  often  been  determined 
either  at  100°  or  at  37.8°  (100°  F.).  The  standard  recently 
established  for  the  United  States  prescribes  a  minimum  specific 
gravity  at  40°,  water  at  the  same  temperature  being  taken  as 
unity.  Either  a  specific  gravity  flask  or  an  Ostwald  pyknom- 
eter  can  be  used  conveniently  for  the  determination,  the 
pyknometer  being  filled  and  adjusted  while  surrounded  by  water 
kept  at  the  required  temperature,  then  removed  from  the  water 
bath,  wiped  dry  on  the  outside,  allowed  to  cool  to  the  tempera- 
ture of  the  balance,  and  weighed. 

Saponification  Number 

This  is  determined  as  described  in  Chapter  VIII.  Since  the 
normal  saponification  numbers  of  butter  fat  are  only  about  15 
per  cent  in  excess  of  those  of  the  fats  commonly  used  as  adulter- 
ants, the  determination  in  order  to  be  of  much  value  must  be 
very  accurately  made. 

Insoluble  Fatty  Acids  —  Hehner  Number 

Reagents.  —  1.  The  alcoholic  potash  solution  used  in  the  de- 
termination of  the  saponification  number. 

2.  Alcohol,  about  95  per  cent  by  volume,  which  leaves  no 
appreciable  residue  upon  evaporation. 

Determination.  —  Saponify  4  grams  of  butter  fat  with  50  cc. 
of  the  alcoholic  potash  solution,  evaporate  to  a  paste  to  expel 
alcohol,  dissolve  the  soap  in  about  400  cc.  of  hot  water  in  a 
weighed  beaker,  add  hydrochloric  acid  in  excess  of  the  amount 
required  to  neutralize  the  potash  used,  and  heat  nearly  to  boil- 
ing with  occasional  stirring  until  the  fatty  acids  have  collected 
in  a  clear  layer  on  the  surface.  Cool  thoroughly,  pour  the  so- 
lution through  a  filter,  and  wash  the  cake  with  cold  water  with- 


192  METHODS   OF   ORGANIC   ANALYSIS 

out  removing  it  from  the  beaker.  Stir  up  the  fatty  acids  in 
the  beaker  with  another  portion  of  hot  water  (200  to  300  cc.), 
cool  thoroughly,  filter,  and  wash  again.  Repeat  this  treatment 
three  times.  After  a  final  thorough  washing  with  cold  water, 
put  the  beaker  containing  the  fatty  acids  beneath  the  funnel 
and  dissolve  any  fatty  acids  which  the  filter  may  contain  by 
washing  with  hot  95  per  cent  alcohol,  allowing  the  washings  to 
run  into  the  beaker.  Evaporate  off  the  alcohol  and  dry  the 
beaker  containing  the  fatty  acids  to  constant  weight  in  a  boiling 
water  oven. 

Notes.  —  This  is  the  modification  of  Hehner's  method  adopted 
by  the  Association  of  Official  Agricultural  Chemists  l  and  by 
the  chemists  of  the  Government  Laboratory,  London.2  The 
original  method,3  which  is  still  largely  used,  involves  washing 
the  melted  fatty  acids  with  hot  water  on  a  paper  filter.  The 
results  thus  obtained  are  usually  1  to  2  per  cent  lower  than 
those  by  the  official  method.  In  the  hot  filtration  method 
there  is  danger  of  washing  some  of  the  melted  fatty  acid 
through  the  paper. 

Iodine  Number 

The  determination  of  the  iodine  number  has  been  fully  de- 
scribed in  Chapter  VIII.  As  butter  fat  absorbs  only  26  to 
38  per  cent  of  its  weight  of  iodine,  a  gram  of  sample  can  be 
used  for  each  determination.  According  to  Patrick,*  butter 
fat  shows  iodine  numbers  about  1  unit  higher  by  the  Hanus 
than  by  the  Hiibl  method. 

Melting  Point  —  Wiley's  Method  5 

Apparatus  and  Reagents.  —  1.  An  accurate  thermometer  read- 
ing to  0.1  degree. 

1  Bui.  107,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 

2  Thorpe  :  J.  Chem.  Soc.,  1904,  85,  248. 

3  Hehner  and  Angell :  Butter,  its  Composition  and  Adulterations.    London, 
1874.     Hehner :  Z.  anal.  Chem.,  1877,  16,  145.     Ephraim,   loc.  cit. 

4  U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  81,  p.  49. 

5  Wiley's  Agricultural  Analysis,  Vol.  Ill ;  Bui.  107,  Bur.  Chem.,  U.  S.  Dept. 
Agriculture. 


EDIBLE    OILS   AND    FATS  193 

2.  A  tall  beaker  nearly  filled  with  water  and  arranged  to 
be  heated   gradually  with  constant  stirring   from   bottom  to 
top. 

3.  A  wide  test  tube  suspended  in  the  water  in  the  beaker 
and  nearly  filled  with  water  and  alcohol  as  follows :   Half  fill 
the  tube  with  hot  recently  boiled  distilled  water,  then  pour  a 
nearly  equal  volume  of   hot   recently  boiled  alcohol  into  the 
tube,  carefully  floating  the  alcohol  on  the  water  with  as  little 
mixing  of  the  liquids  as  possible. 

Determination.  —  Allow  a  drop  of  the  melted  butter  fat  to 
fall  upon  a  smooth  piece  of  ice  floating  in  recently  boiled  dis- 
tilled water.  A  thin  disk  of  fat  about  1  cm.  in  diameter  should 
be  obtained.  Remove  the  disk  from  the  ice  by  forcing  the 
latter  below  the  water  when  the  fat  will  come  to  the  surface 
whence  it  is  removed  by  means  of  a  steel  spatula  or  knife  blade 
and  dropped  into  the  tube  containing  the  water  and  alcohol. 
The  disk  sinks  to  the  point  where  the  density  of  the  alcohol- 
water  mixture  is  equal  to  its  own.  It  must  not  touch  the  side 
of  the  tube.  Suspend  the  thermometer  so  that  the  bulb  hangs 
in  the  tube  exactly  level  with  the  disk  of  fat.  Gradually  heat 
the  beaker,  keeping  the  water  well  stirred.  After  the  disk 
begins  to  shrivel,  indicating  that  the  temperature  is  within  a 
few  degrees  of  the  melting  point,  the  heat  must  be  applied 
very  carefully.  The  temperature  at  which  the  fat  becomes  a 
sphere  is  taken  as  the  melting  point.  Repeat  the  determina- 
tion twice,  heating  the  bath  at  such  a  rate  that  8  to  10  minutes 
are  required  to  raise  the  temperature  through  the  last  2 
degrees.  The  second  and  third  determinations  should  agree 
within  0.2°. 

Notes.  —  The  special  -advantage  of  this  method  is  that  it 
avoids  the  discrepancies  caused  by  the  adherence  of  the  melting 
fat  to  solid  surfaces,  which  in  most  other  methods  makes  it 
difficult  to  determine  the  exact  point  of  fusion.  It  is  important 
to  secure  a  very  thin  disk  of  fat  for  the  determination,  to  avoid 
all  adherence  of  air  bubbles,  and  to  secure  uniform  heating  of 
the  thermometer  bulb  and  the  disk  by  occasionally  swaying  the 
former  around  the  latter  as  the  temperature  approaches  the 


194  METHODS   OF   ORGANIC   ANALYSIS 

melting  point.  By  using  hot  recently  boiled  water  and 
alcohol  in  preparing  the  test  tube  for  the  determination  the 
danger  of  air  bubbles  is  avoided. 

Additional  Determinations 

The  index  of  refraction  and  Crismer's  test1  based  upon  the 
critical  temperature  of  solution  in  alcohol  are  considerably  used 
in  the  examination  of  butter  fat.  They  are  especially  useful 
where  rapid  "  sorting  tests  "  are  required,  as  in  food  inspection 
laboratories,  where  only  the  suspected  samples  are  submitted  to 
complete  examination. 

The  phytosteryl  acetate  test  2  is  occasionally  employed  as  a 
means  of  detecting  the  presence  of  vegetable  fat,  but  requires 
too  much  time  and  skill  for  routine  use  and  is  liable  to  give 
misleading  results  in  the  hands  of  an  inexperienced  person. 

The  chief  vegetable  fats  in  use  as  butter  substitutes  are  cot- 
tonseed oil,  the  methods  for  which  have  already  been  described, 
and  coconut  oil,  whose  detection  is  discussed  below.  (See  also 
the  references  at  the  end  of  this  chapter.) 

Acidity  of  butter  fat  is  sometimes  determined  and  interpreted 
as  a  measure  of  the  rancidity,  although  the  odor  and  taste  which 
cause  a  butter  to  be  regarded  as  rancid  are  more  largely  due  to 
aldehydes  and  other  decomposition  products  than  to  free  fatty 
acids.  On  the  assumption  that  acidity  can  serve  as  a  measure 
of  rancidity,  the  term  degree  of  rancidity  is  sometimes  used  as 
synonymous  with  degree  of  acidity,  i.e.  to  show  the  number  of 
cubic  centimeters  of  normal  alkali  required  to  neutralize  the 
free  acid  in  100  grams  of  fat.  One  "  degree  "  is,  therefore, 
equivalent  to  an  acid  number  of  0.56  or  to  0.28  per  cent  of  free 
oleic  acid. 

iCrismer:  Bull.  Assoc.  beige.  Chim.,  1895,  9,  71;  1896,  10,312;  Abs. 
Analyst.,  1895,  20,  209;  1897,  22,  71.  See  also  Weiss:  Pharm.  Ztg.,  41, 
268;  Chem.  CentrbL,  1896,  I,  1212.  Asboth :  Chem.  Ztg.,  1896,  20,  685. 
Browne:  J.  Am.  Chem.  Soc.,  1899,  21,  990. 

2  Bomer  :  Z.  Nahr.-Genussm.,  1901,  4,  865,  1070  ;  1902,  5,  1018.  Juckenack 
and  Pasternack  :  Ibid.,  1904,  7,  193.  Gill  and  Tufts  :  J.  Am.  Chem.  Soc.,  25, 
251,  254,  498.  Tolman  :  J.  Am.  Chem.  Soc.,  27,  589.  Lewkowitsch:  Oils, 
Fats,  and  Waxes,  4th  Ed.,  p.  473. 


EDIBLE  OILS  AND  FATS 
COMPOSITION  OF  BUTTER  FAT 


195. 


Browne  analyzed  the  mixture  of  fatty  acids  from  a  sample 
having  a  rather  low  iodine  number  (29.28)  with  the  following 
results  : 1 


Acids 

Percentage  of  acid 
in  fat 

Corresponding  per- 
centage of  tri- 
glyceride 

Oleic  

32.50 

33.95 

Dioxystetiric    

1.00 

1  04 

Ste&ric                        .     .          

1.83 

1  91 

Palmitic 

3861 

40  51 

9.89 

10.44 

Laurie     

2.57 

2.73 

Capric         ....          

032 

034 

C  a,  pry  lie                                               .     .     . 

049 

053 

Caproic 

2  09 

2  39 

Butyric  

5.45 

6.23 

Total   

94.75 

100  00 

This  calculation  neglects  the  unsaponifiable  matter,  which 
according  to  Browne  amounts  to  only  about  0.1  per  cent. 

The  composition  of  butter  fat  is,  however,  quite  variable,  as 
will  be  seen  from  the  range  in  analytical  properties. 

VARIATIONS  AND  RELATIONS  OF  ANALYTICAL  PROPERTIES 

The  Reichert-Meissl  or  Reichert-Wollny  number  is  much 
the  most  important  of  the  data  obtained  in  the  examination  of 
butter  fat.  The  proportion  of  volatile  acids  tends  to  decrease 
as  the  period  of  lactation  advances.  The  estimated  normal 
range  for  the  other  important  properties  has  been  given  in  the 
table  at  the  end  of  Chapter  VIII. 

Any  of  these  properties  may  be  influenced  by  the  feeding  or 
health  of  the  animal  and  occasionally  vary  much  beyond  the 
usually  accepted  "  normal "  limits,  as  is  shown  by  the  following 
data  collected  by  Browne:  2 

1  J.  Am.  Chem.  Soc.,  1899,  21,  823. 

2  J.  Am.  Chem.  Soc.,  1899,  21,  632. 


196 


METHODS  OF  ORGANIC  ANALYSIS 


General 
limits 

Extreme  limits 

Reichert-Meissl  number 
Saponification  number  .     . 

Iodine  number  

20-33 
220-236 

26-38 

11.2[Morse]-41  [Nilson] 
216  [Samelson]-245[Fischer] 
19.5  [Moore]  -49.57    f  Farnsteiner  "I 

\_  and  Karsch  J 

The  results  of  analyses  of  357  authentic  samples  of  butter 
fat  collected  from  various  parts  of  Great  Britain  and  examined 
at  the  Government  Laboratory,  London,  have  been  arranged 
by  Thorpe l  according  to  the  Reichert-Wollny  numbers  and 
averaged  by  groups  to  show  the  relations  between  the  principal 
physical  and  chemical  properties  of  pure  butter  fat. 

The  following  table  shows  the  averages  for  each  group  of 
samples.  The  first  line,  for  example',  gives  the  average 
Reichert-Wollny  number  for  all  samples  in  which  this  number 
lay  between  22.00  and  22.99,  with  the  average  of  the  same 
samples  for  each  of  the  other  determinations.  The  second 
line  shows  the  averages  for  all  samples  having  Reichert-Wollny 
numbers  from  23.00  to  23.99,  etc. 

TABLE  17.  —  RELATION  OF  PHYSICAL  AND  CHEMICAL  PROPERTIES  OF 
BUTTER  FAT  (THORPE) 


Number 
of 
samples 

Reichert- 
Wollny 
number 

Specific 
gravity 
37.8° 
37.  b° 

Butyro- 
refractometer 
reading 

at  45° 

Saponification 
number 

Insoluble 
fatty  acids 
per  cent 

Mean 
molecular 
weight  of 
insoluble 
acids 

7 

22.5 

0.9101 

42.0 

219.9 

90.1 

266.9 

17 

23.5 

0.9104 

41.5 

221.6 

89.7 

265.5 

15 

24.5 

0.9108 

41.5 

223.5 

89.4 

265.0 

27 

25.5 

0.9110 

41.3 

223.6 

89.3 

264.2 

37 

26.5 

0.9113 

41.0 

225.6 

88.9 

261.9 

51 

27.5 

0.9114 

40.6 

227.0 

88.7 

261.7 

78 

28.8 

0.9118 

40.1 

228.6 

88.4 

260.9 

56 

29.5 

.    0.9120 

40.1 

230.2 

88.3 

259.6 

41 

30.5 

0.9123 

39.9 

231.7 

87.9 

260.1 

18 

31.3 

0.9125 

39.7 

232.5 

87.9 

258.0 

10 

32.6 

0.9130 

39.4 

232.8 

87.7 

257.8 

1  J.  Chem.  8oc.,  1904,  85,  248. 


EDIBLE    OILS   AND    FATS 


197 


In  order  to  show  to  what  extent  increase  of  volatile  acids 
takes  place  at  the  expense  of  oleic  acid,  the  iodine  numbers  of 
50  of  the  above  samples  were  determined.  Arranging  these  in 
groups  of  20  and  30,  respectively,  according  to  the  Reichert- 
Wollny  values,  the  following  average  figures  were  obtained  : 


Reichert- 
Wollny 
number 

Iodine 
number 

Oleic  acid 
per  cent 

Insoluble 
acid 
per  cent 

Mean  molecular 
weight  of 
insoluble  acids 

First  group       

04  9 

40.0 

44.4 

89.6 

264.6 

Second  jjroup             •     .     . 

30.8 

32.4 

36.0 

88.1 

259.8 

DETECTION  OF  OLEOMARGARINE 

Butter  substitutes  or  "artificial  butters,"  unless  sold  under 
special  names  indicating  their  origin,  are  collectively  termed 
"  oleomargarine "  in  America  or  "  margarine "  in  England. 
The  oleomargarine  made  in  America l  consists  chiefly  of  refined 

1  The  materials  used  in  the  manufacture  of  oleomargarine  in  the  United 
States  during  the  fiscal  year  ending  June  30,  1899  (Senate  Document  No.  168, 
57th  Congress,  1st  Session),  were  as  follows  : 

TABLE  18.  —  MATERIALS  USED  IN  MANUFACTURE  OF 
OLEOMARGARINE,  1899 


Material 

Quantity 

Percentage 
of  the 
whole 

Value 
per 
pound 

Total  value 

Neutral  lard      

Pounds 
31,297,251 

34.27 

Cents 

8 

$2,503  780.08 

Oleo  oil              .          .... 

24  491  769 

26.82 

9 

2  144  917  69 

Cottonseed  oil  .     ..... 
"  Butter  oil  "   

4,357,514 
4,342,904 

4.77 
4.76 

6 
6 

522,025.08 
260,520.00 

Sesame  oil   

486,310 

.53 

10 

4,863  10 

Coloring  matter     .... 

148,970 

.16 

20 

29  296  00 

Sugar                      . 

110  164 

.12 

4 

4  406  50 

Glycerin 

8  963 

01 

10 

896  30 

Stearin    

5,890 

.007 

8 

459  60 

Glucose   

2,550 

003 

3 

76  50 

Milk         .     .               ... 

14  200  576 

15  55 

1 

142  005  76 

Salt          .               ... 

6  773  670 

7  42 

1 

67  726  70 

Butter 

1  568  319 

1  72 

20 

313  663  80 

Cream 

3  527  410 

3  86 

5 

176  370  50 

Total     

91,322,260 

100 

$6,171,007.61 

198  METHODS   OF   ORGANIC    ANALYSIS 

lard,  "  oleo  oil "  (the  soft  part  of  beef  fat),  and  cottonseed  oil, 
often  mixed  with  a  small  amount  of  butter  and  almost  always 
churned  with  milk  or  cream.  Palm  oil  and  sesame  oil  are 
known  to  be  used  to  some  extent  and  other  semi-drying  and 
non-drying  oils  are  probably  utilized  in  some  factories. 

Hence,  in  comparison  with  any  of  the  common  constituents  of 
oleomargarine,  butter  fat  is  characterized  by  its  high  proportion 
of  soluble  volatile  acids,  together  with  low  percentages  of  oleic 
and  stearic  acids.  The  presence  of  oleomargarine  in  butter  fat, 
therefore,  lowers  the  Reichert-Meissl  and  saponification  numbers 
and  the  specific  gravity,  while  it  raises  the  percentage  of  in- 
soluble acids  and  either  the  melting  point  or  the  iodine  number 
or  both. 

Several  European  governments  require  that  sesame  oil  be 
added  to  oleomargarine  in  order  to  faciliate  its  detection  when 
mixed  with,  or  substituted  for,  butter-.  Similarly,  the  addition 
of  butter  to  oleomargarine  is  sometimes  forbidden  or  restricted 
in  order  to  prevent  the  production  of  mixtures  too  closely 
resembling  genuine  butter.  The  essential  features  of  the  oleo- 
margarine laws  of  the  principal  countries  are  given  in  Lewko- 
witsch's  Oils,  Fats,  and  Waxes. 

DETECTION  OF  COCONUT  FAT 

Since  coconut  fat  consists  largely  of  glycerides  of  saturated 
acids  of  low  molecular  weight,  it  could  be  added  in  considerable 
quantity  to  butter  fat  of  average  composition  without  causing 
the  latter  to  vary  beyond  the  normal  limits  in  any  of  the  im- 
portant analytical  properties.  A  comparison  of  the  Reichert- 
Meissl  and  saponification  numbers,  however,  would  lead  to  the 
detection  of  this  adulteration  since  the  former  number  is  higher 
and  the  latter  lower  in  butter  than  in  coconut  fat.  In  pure 

"Butter  oil"  is  commonly  stated  to  be  a  special  brand  of  cottonseed  oil ; 
but  the  high  federal  tax  laid  upon  artificially  colored  oleomargarine  by  the  law 
of  May  9,  1902,  practically  prohibits  the  use  of  coloring  matters  employed  before 
that  date  and  has  led  to  the  introduction  of  "butter  oils"  containing  palm  oil 
which  is  naturally  highly  colored  (Crampton  and  Simons  :  J.  Am.  Chem.  Soc  , 
1905,27,270). 


EDIBLE    OILS    AND    FATS  199 

butter  fat  the  value  of  the  factor  [Saponification  number  — 
(200  +  Reichert-Meissl  number)]  varies  from  3.4  to  —  4.1;  in 
pure  coconut  fat  it  varies  from  47  to  50. 7. * 

Another  method  of  showing  the  presence  of  coconut  oil  is  to 
determine  the  volume  of  tenth-normal  alkali  required  to  neu- 
tralize the  insoluble  volatile  acids  from  5  grams  of  fat.  Under 
the  conditions  described  by  Polenske  2  the  results  thus  obtained 
are  approximately  quantitative,  the  percentage  of  insoluble 
volatile  acids  (mainly  lauric  acid)  being  much  higher  in  coco- 
nut fat  than  in  butter. 

REFERENCES 
I 

(See  book  references  at  end  of  preceding  chapter.) 

II 

1887-89.   WILEY:  Butter  and  Lard,  Parts  1  and  4,  Bui.  13,  Bur.  Chem., 
U.  S.  Dept.  Agriculture. 

1897.  SADTLER  :  Arachis  (Peanut)  Oil.     Am.  J.  Pharm.,  69,  490 ;  Analyst, 

22,  284. 

1898.  ARCHBUTT  :  Estimation  of  Arachidic  Acid.     J.  Soc.  Chem.  Ind.,  17, 

1124. 

HOPKINS:  Maize  Oil.     J.  Am.  Chem.  Soc.,  20,  948. 
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Chem.,  1898,  464. 
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46,  705. 

1899.  BELLIER:   Color  Reactions  for  Sesame   Oil.     Ann.  de  Chim.  Anal., 

4,  217;  Analyst,  25,  50. 

BROWNE  :  Butter  Fat.     J.  Am.  Chem  Soc.,  21,  632,  823,  975. 
COCHRAN  :  Butter  and  Butter  Adulterants.     J.  Franklin  Inst.,  147,  85. 

1900.  BELLIER  :  Detection  of  Arachis  Oil.     Bull.  Soc.  Chim.,  [3],  23,  358. 
ESTCOURT  :  Butters   from   Various   Countries   Compared.     Analyst, 

25,  113. 

OILAR  :  Halphen  Test.     Am.  Chem.  J.,  24,  355. 
WILLIAMS  :  Maize  Oil.     Analyst,  25,  146. 

1  Juckenack  and  Posternack  :  Z.  Nahr.  Genussm.,  1904,  7,  193. 

2  Polenske:  Ibid.,  1904,7,  273.     See   also,   Lewkowitsch's  Oils,    Fats,  and 
Waxes.    Muntz  and  Coudon :  Ann.  d.  Inst.  Agron.,  1904;  Analyst,  1905,  30, 
155.   Hesse  :  Milchwirthschaftl.  Centrbl,  1905,  1,  13  ;   Chem.  Centrbl.,  1905,   I, 
566. 


200  METHODS   OF   ORGANIC    ANALYSIS 

1900-01.   VULTE  and  GIBSON  :  Maize  Oil.    «/.  Am.  Chem.  Soc.,  22,  453  ;  23, 1. 

1901.  HOLM,  KRARUP,  and   PETERSON:   Refraction,  Iodine  Number,  and 

Volatile  Acid  Content  of  Butter.     Z.  Nahr.  Genussm.  4,  746. 

PFRRIN  :  Separation  of  Arachidic  Acid.  Monat.  scientif.,  [4],  15,  320; 
Z.  Nahr.  Genussm.,  4,  986. 

SIEGFELD  :  Judgment  of  Butter  by  Reichert-Meissl  Number.  Z. 
Nahr.  Genussm.,  4,  433. 

WEEMS  and  GRETTENBERG  :  Analytical  Characters  of  Different  Cot- 
tonseed Oils.     Proc.  Iowa  Acad.  Sci.,  1901 ;  Z.  Nahr.  Genuxsm., 
1902,  5,  465. 
1901-02.   BOMER:  Phytosterol  Test.   Z.Nahr.  Genussm,,  4,  865, 1070;  5, 1018. 

1902.  BEHREND  and  WOLFS  :  Butter  Fats  from  Individual  Cows.     Z.  Nahr. 

Genussm.,  5,  689. 

FULMER  :  Halphen's  Test.     J.  Am.  Chem.  Soc.,  24,  1148. 
LAXA  :  Change  of  Butter  Fat  by  Microorganisms.     Arch.  Hyg.,  41, 

119. 
RITTER  :  Phytosterol.     Z.  physiol.  Chem.,  34,  430,  461 ;  35,  550. 

1902.  TOLMAN  and  MUNSON  :  Olive  Oil  and  its  Substitutes.    Bui.  77,  Bur. 

Chem.,  U.  S.  Dept.  Agriculture. 

1903.  CRAMPTON:  Composition  of  Process  or  Renovated  Butter.     J.  Am. 

Chem.  Soc.,  25,  358. 
GILL  and  TUFTS  :  (Cholesterol,    Phytosterol,    Sytosterol.)      J.    Am. 

Chem.  Soc.,  25,  251,  254,  498. 

KREIS  :  Detection  of  Sesame  Oil.     Chem.  Ztg.,  27,  1030. 
SWAYING  :  Influence  of  Feeding  Cottonseed  and  Sesame  Meal  on  the 

Properties  of  Butter  Fat.     Z.  Nahr.  Genussm.,  6,  97. 
TOLMAN  and  MUNSON  :  (Analysis  of  Salad  Oils).    J.  Am.  Chem.  Soc., 

25,  954. 
Wus  :  (Iodine  Numbers  of  Edible  Oils).    Z.  Nahr.  Genussm.,  6,  692. 

1904.  GROSSMANN  arid  MEINHARD  :  Dutch  Butter.     Z.  Nahr.   Genussm., 

3,  237. 

KRAUS:  (Conditions  Affecting  the  Keeping  Qualities  of  Butter). 
Arb.  kaiserl.  Gesundheitsamte,  22,  235 ;  Z.  Nahr.  Genussm.,  9,  286. 

1905.  ARNOLD:  (Analysis  of  Food  Fats).     Z.  Nahr.  Genussm.,  10,  201. 
CRAMPTON  and  SIMONS  :  Detection  of  Palm  Oil  when  used  as  a  Color- 
ing Material  in  Oils  and  Fats.    J.  Am.  Chem.  Soc.,  27,  270. 

FISCHER  and  PEYAN:  (Detection  of  Cottonseed  Oil).  Z.  Nahr. 
Genussm.,  9,  81. 

LYTHGOE  :  Refractive  Index  of  Codliver  Oil.  J.  Am.  Chem.  Soc.,  27, 
887. 

PARRY:  Codliver  Oil  Standards.  Chemist  and  Druggist,  46,  491; 
Analyst,  30,  208. 

POLENSKE:  (Analysis  of  Lard  and  Butter).  Arb.  kaiserl.  Gesund- 
heitsamte, 22,  557,  576;  Analyst,  31,  46. 


EDIBLE   OILS   AND   FATS  201 

1905.  TOLMAN:  Phytosteryl  Acetate  Test  for  Examination  of  Lard  from 

Cottonseed -meal-fed  Hogs.     /.  Am.  Chem.  Soc.,  27,  589. 
WESSON  and  LANE  :  Quantitative  Analysis  of  Lard.     /.  Soc.  Chem. 
Ind.,  24,  714. 

1906.  ASCHMANN  and  AREND  :  Determination  of   Water   in   Butter   and 

Other  Fats.     Chem.  Ztg.,  30,  953. 
FARNSTEINER  :  (Examination  of  Lard).     Z.  Nahr.  Genussm.,  11,  1 ; 

Analyst,  31,  72. 

HARRIS  :  Estimation  of  Coconut  Oil  in  Butter  Fat.     Analyst,  31,  353. 
PATRICK:  Rapid  Determination  of  Water  in  Butter.     J.  Am.  Chem. 

Soc.,  28,  1611. 
EJDEAL  and  HARRISON:  On  the  Polenske  Method  for  Coconut  Oil 

in  Butter.     Analyst,  31,  254. 

TOLMAN:  American  Codliver  Oils.     J.  Am.  Chem.  Soc.,  28,  388. 
WALKER:  Coconut  Oil.     Philippine  J.  Sci.,  1,  117;  Analyst,  31,  165. 

1907.  AMBERGER:  Influence  of  Food  on  Composition  of  Butter  Fat.     Z. 

Nahr.  Genussm.,  13,  614. 
ARCHBUTT  :    Tunisian  and  Algerian  Olive  Oils.     J.  Soc.  Chem.  Ind., 

26,  453. 
BELLIER  :   Analysis  of  Butter  (New  Method).     Ann.  de  Chim.  Anal., 

11,  412  ;  Analyst,  32,  22. 

HANUS:    (Detection  of  Coconut  Oil).     Z.  Nahr.  Genussm.,  13,  18. 
HENSEVAL  and  HUVART  :   Contribution  to  the  Study  of  Fish  Liver 

011.  Chem.  Rev.  Fette-  Harz-Ind.,  14,  191;  Chem.  Abs.,  1,  2751. 
HINKS  :   Detection  of  Coconut  Oil  in  Butter.     Analyst,  32,  160. 
HODGSON  :  Detection  of  Coconut  Oil  in  Butter.     Chem.  News,  95, 121. 
JEAN:   Examination  of  Butter.     Rev.   gen.  Chim.,   10,  253;  Chem. 

Abs.,  1,  2617. 
KUHN  and  BENGEN  :  Cause  of  Halphen  Reaction.     Z.  Nahr.  Genussm., 

12,  145. 

KUHN  and  HALEPAAP:  Critical  Study  of  Welman's  Reaction.  Z. 
Nahr.  Genussm.,  12,  449. 

MCPHERSON  and  RUTH  :  Corn  Oil  —  Its  Possibilities  as  an  Adulter- 
ant in  Lard  and  its  Detection.  J.  Am.  Chem.  Soc.,  29,  921. 

PATRICK:  Rapid  Determination  of  Water  in  Butter.  J.  Am.  Chem. 
Soc.,  29,  1126. 

SIEGFELD  :  (Detailed  Study  of  Polenske  Number) .   Chem.  Ztg.,  32,  511. 

:  Influence  of  Feed  on  Butter  Fat.     Z.  Nahr.  Genussm.,  13,  513: 

SMITH  :  Application  of  Arachidic  Acid  Test  to  Solid  Fats.  J.  Am. 
Chem.  Soc.,  29,  1756. 

SOLTSEIN  :  Detection  of  Tallow  and  Lard  in  Presence  of  Each 
Other.  Chem.  Rev.  Fette-Harz-lnd.,  13,  240;  Chem.  Abs.,  1,  109. 

WINDAUS  :  Separation  of  Cholesterol  and  Phytosterol.  Chem.  Ztg., 
30,  1011. 


202  METHODS  OF  ORGANIC  ANALYSIS 

1908.  CORNELISON  :   Detection  of  Synthetic  Color  in  Butter.     J.  Am.  Chem. 

Soc.,  30,  1478. 
EMERY:   Detection  of  Beef  Fat  in  Lard.     U.  S.   Dept.   Agr.,   Bur. 

Animal  Ind.,  Cir.  132  ;  Chem.  Abs.,  2,  2461. 
FARRINGTON  :   Determination  of  Water  in  Butter.     Wis.  Agl.  Expt. 

Station,  Bui.  154 ;  Chem.  Abs.,  2,  870. 

FULMER  and  MANCHESTER  :   Effect  of  Heat  on  Cottonseed  Oil  Con- 
stants.    /.  Am.  Chem.  Soc.,  30,  1477. 
FRITSCHE:    (Polenske     Number     of      Dutch     Butter).      Z.     Nahr. 

Genussm.,  15,  193. 
HANUS  and  STEHL  :   The  Ethyl  Ester  Number,  A  New  Method  for 

Coconut  Fat.     Z.  Nahr.  Genussm.,  15,  576. 
KREIS  :   Influence  of  Rancidity  on  Baudouin  Reaction.     Chem.  Abs., 

2,  2165,  2166. 
WAGNER  and  CLEMENT  :   Cottonseed  Oil.     Z.  Nahr.  Genussm.,  16, 

145. 

1909.  KOHNIG  and  SCHLUCKEBIER  :   Influence  of  Fat  in  Feed  upon  Fat  of 

Pigs  with  Special  Regard  to  Phytosterol.     Z.  Nahr.  Genussm., 

15,  641. 
TATLOCK  and  THOMSON  :   The  Value  of  the  Polenske  Method.     J. 

Soc.  Chem.  Ind.,  28,  69. 
MILLIAU  :    (Reactions  of  Kapok  and  Baobab  Oils).     Matieres  grasses, 

2,  1545  ;   Chem.  Abs.,  4,  969. 

1910.  BULL  and  SAETHER  :    Can  One  Determine  the  Nature  of  the  Vege- 

table Oil  on  Sardines?     Chem.  Ztg.,  34,  733. 
GIBBS   and  AGCAOILI:   Lard  from  Wild  and  Domestic   Philippine 

Hogs    and    the    Changes    Effected    by   Feeding    Copra    Cake. 

Philippine.  J.  Sci.,  5A,  33;   Chem.  Abs.,  4,  2748. 
HARE  :   Some   Effects  of  Feeds   upon   the  Properties  of  Lards.     /. 

Ind.  Eng.  Chem.,  2,  264. 
LINDSAY  ET  AL:    (Effect  of  Feed  on  Butter  Fat).     Mass.  Agl.  Expt. 

Sta.  Report,  1908,  66-110;   Chem.  Abs.,  4,  1774. 

1911.  ARNOLD:   Determination  of  Coconut  Oil  in  Edible  Fats.     Z.  Nahr. 

Genussm.,  21,  587. 
MARCILLE:   Olive   Oils   from   Tunis.     Ann.  falsif.,   3,   372;   Chem. 

Abs.,  5,  732. 
REVIS  and  BOLTON  :   Methods  of  Estimating  Coconut  Oil  and  Butter 

in  Butter  and  Margarine.     Analyst,  36,  333. 
RICHARDSON  :   Coconut   Oil   of    High    Iodine   Value.     /.  Ind.  Eng. 

Chem.,  3,  574. 
STEENBOCK  :   Modification  of  Wiley's  Method  for  Melting  Point  of 

Fats.     /.  Ind.  Eng.  Chem.,  2,  480. 
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Ann.  falsif.,  4,  139;   Chem.  Abs.,  5,  1947. 


CHAPTER   X 
Drying  Oils 

SEVERAL  oils  of  both  vegetable  and  animal  origin  (among  the 
latter  more  particularly  the  fish  oils)  contain  glycerides  of 
highly  unsaturated  fatty  acids  which  on  exposure  in  thin  layers 
to  air  readily  absorb  oxygen  and  become  converted  into  solids. 
This  property  gives  rise  to  the  term  "  drying  "  oils.  A  few  of 
the  more  prominent  drying  oils  may  be  listed  as  follows  : 

VEGETABLE  DRYING  OILS 

Linseed  oil,  pressed  from  the  seeds  of  the  flax  plant,  pro- 
duced in  large  quantities  in  North  and  South  America,  Russia, 
and  India,  is  the  most  important  of  the  drying  oils  and  is  ex- 
tensively used  for  direct  application  as  a  protective  coating  to 
wood  and  metal,  and  in  the  manufacture  of  paints  and  varnishes, 
oilcloths,  linoleum,  printing  inks,  rubber  substitutes,  etc. 
Lewkowitsch  estimates  that  there  were  produced  in  1907, 
1,200,000  tons  of  linseed  in  Argentina ;  646,275  tons  in  the 
United  States;  450,000  tons  in  Russia;  and  419,900  tons  in 
India.  Linseed  yields  in  general  about  35  per  cent  of  its  weight 
of  oil. 

The  seed  are  usually  crushed  between  rollers,  then  heated  to 
about  160°  F.  and  pressed  while  warm.  The  oil  so  obtained  is 
yellowish  brown  or  brownish  yellow  and  somewhat  turbid  from 
traces  of  moisture  and  mucilaginous  material,  which  impurities, 
however,  gradually  settle  out  when  the  oil  is  stored.  Oil 
which  has  been  thus  purified  by  long  standing  is  sometimes 
called  "tanked  oil."  A  more  rapid  method  of  refining  is  to 

203 


204  METHODS   OF   ORGANIC    ANALYSIS 

treat  the  oil  with  1  to  2  per  cent  of  sulphuric  acid.  This  pro- 
duces a  charred  mass  which  carries  down  with  it  the  greater 
part  of  the  impurities. 

Boiled  linseed  oils  are  made  by  heating  linseed  oil  with  a 
"drier,"  formerly  to  temperatures  of  210°  to  260°  C.,  now, 
according  to  Lewkowitsch,  to  a  temperature  of  about  150°  C. 
The  extent  to  which  the  analytical  characters  are  changed 
depends  upon  the  details  of  the  process  and  the  extent  to  which 
the  oil  is  exposed  to  air. 

Tung  oil,  also  called  Chinese  or  Japanese  wood  oil,  is  pressed 
from  the  nuts  of  the  tung  tree.  According  to  Ennis  the  use 
of  this  oil  in  the  United  States  is  steadily  increasing  and  experi- 
ments are  being  made  with  the  cultivation  of  the  tree  in  Cali- 
fornia. At  present  the  oil  comes  chiefly  from  China,  where  the 
natives  roast  and  crush  the  nuts  and  press  from  them  about 
40  per  cent  of  oil,  which  is  only  about  three  fourths  of  the 
amount  present.  According  to  Lewkowitsch  the  oil  is  also 
produced  in  Madagascar  under  the  name  of  "  Bakoly  oil." 
Tung  oil  has  a  characteristic  persistent  odor  which  is  not  easily 
removed  by  refining.  It  is  used  largely  for  oiling  wood  and 
waterproofing  paper.  Lewkowitsch  states  that  while  many 
patents  have  been  taken  out  for  the  substitution  of  tung  oil  for 
linseed  oil  in  manufactures,  little  progress  has  yet  been  made  in 
this  direction. 

Walnut  oil  and  poppyseed  oil,  while  they  have  not  such 
pronounced  drying  properties  as  linseed  and  tung  oils, 
have  the  advantage  of  yielding  almost  colorless  films  which 
are  not  likely  to  crack.  These  oils  are  therefore  especially 
adapted  to  use  in  white  or  delicately  colored  paints  for 
artists. 

Maize  oil  has  not  sufficient  drying  property  to  be  useful  as 
a  paint  oil  but  "  dries  "  well  enough  to  permit  of  its  use  in  putty. 
It  is  also  used  like  linseed  oil  in  making  rubber  substitute.  It 
has  probably  been  used  to  some  extent  as  an  adulterant  of  lin- 
seed oil. 

Soy  (soja,  soya)  bean  oil  is  used  to  some  extent  in  admixture 
with  linseed  oil. 


DRYING   OILS  205 

FISH  OILS 

In  recent  years  the  fish  oil  industry  has  been  much  altered 
through  the  introduction  of  steam  trawlers  and  the  prompt 
rendering  of  the  oil,  which  results  in  a  product  largely  free  from 
the  dark  color  and  rank  odor  formerly  regarded  as  characteristic 
of  fish  oils. 

Menhaden  oil  is  obtained  from  the  bodies  of  the  menhaden,  a 
fish  somewhat  larger  than  a  herring,  which  abounds  in  the 
Atlantic  especially  off  the  coast  of  New  Jersey. 

Toch  1  states  that  menhaden  is  the  best  fish  oil  for  use  in 
paint  and  that  the  winter-bleached  variety  is  to  be  preferred. 
This  should  be  fairly  pale  in  color,  with  an  iodine  number  of 
150  or  over,  and  with  little  or  no  fishy  odor.  Such  an  oil, 
Toch  finds,  may  be  mixed  with  linseed  oil  even  up  to  75  per 
cent  of  the  mixture  with  good  results  when  used  for  exterior 
painting  where  its  odor  is  not  noticeable.  In  fact  paint  made 
from  such  a  fish  oil  is  said  to  be  more  resistant  to  heat  than  a 
linseed  oil  paint  and  therefore  preferable  for  smoke  stacks, 
boiler  fronts,  etc.  It  is  also  said  to  be  more  resistant  to  the 
salt  air  of  the  seacoast.  Menhaden  oil  has  also  been  used  in 
place  of  linseed  oil  in  the  manufacture  of  enamel  leather  and  of 
printing  inks. 

Japanese  sardine  oil,  sardine  oil,  and  herring  oil  have  some 
drying  property  and  are  used  to  some  extent  as  partial  sub- 
stitutes for,  or  adulterants  of,  linseed  oil. 

LINSEED  OIL 

ANALYTICAL   PROPERTIES   OF   LINSEED   OIL 

Commercial  linseed  oil  is  usually  designated  by  the  region  of 
its  origin.  It  varies  considerably,  the  variation  being  due  mainly 
to  the  presence  of  foreign  seeds  in  the  linseed  at  the  time  of 
pressing.  Hempseed  is  practically  always  present,  sometimes 
in  very  small  proportions  but  often  to  the  extent  of  5  per  cent, 
or  more,  of  the  weight  of  seed.  The  drying  properties  of  lin- 

1J.  Ind.  Eng.  Chem.,  3,  "627. 


206  METHODS    OF   ORGANIC    ANALYSIS 

seed  oil  are  better  the  purer  the  seeds  from  which  it  is  pressed. 
The  iodine  number  and  the  drying  power  of  the  oil  decrease  as 
the  proportion  of  hempseed  increases.  Hence  a  linseed  oil  con- 
taining much  hemp  oil  wo.uld  be  shown  to  be  of  inferior  quality 
by  its  low  iodine  number,  but  could  not  be  pronounced  adulterated 
so  long  as  this  did  not  fall  below  170.  The  maximum  iodine 
number  of  linseed  oil  is  difficult  to  fix,  since  the  results  obtained 
by  the  method  of  Wijs  often  exceed  the  Hubl  numbers,  but  since 
no  other  common  oil  has  a  higher  iodine  number  than  linseed, 
the  maximum  limit  is  of  little  practical  importance  in  the  detec- 
tion of  adulterations. 

The  usual  range  of  the  more  important  analytical  constants 
of  linseed  oil  has  been  given  in  the  table  at  the  end  of  Chap- 
ter VIII.  The  interpretation  of  "  constants,"  their  relations  to 
each  other,  and  their  use  in  the  detection  of  adulterations  having 
been  discussed  in  some  detail  in  connection  with  the  exam- 
ination of  salad  oils,  it  will  be  sufficient  in  this  case  to  mention 
briefly  the  principal  adulterants  with  means  for  the  detection 
of  each,  and  describe  the  hexabromide  test  which  distinguishes 
linseed  from  practically  all  other  oils. 

ADULTERANTS   AND   METHODS   OF  DETECTION 

Mineral  Oil 

Mineral  oil  would  greatly  lower  the  iodine  number,  tempera- 
ture reaction,  and  saponification  number.  Whenever  a  low 
saponification  number  is  found,  the  unsaponifiable  matter  should 
be  separated  and  examined.  Any  mineral  oil  which  is  not 
volatile  at  100°  can  be  separated  quantitatively,  dried,  and 
weighed.  Volatile  mineral  oil  can  be  distilled  by  means  of  a 
current  of  steam,  separated  from  water  in  the  distillate,  and 
measured  or  weighed.  In  case  turpentine  were  present,  as  in 
some  so-called  "  boiled "  oils,  it  would  be  distilled  with  steam 
and  would  separate  from  water  in  the  distillate  in  the  same 
way.  The  optical  rotatory  power  of  turpentine  affords  an 
easy  means  of  distinguishing  it  from  benzine  or  other  volatile 
mineral  oil. 


DRYING   OILS  207 

Rosin  and  Rosin   Oil 

Rosin  dissolved  in  linseed  oil  raises  the  specific  gravity  and 
index  of  refraction  while  the  saponification  and  iodine  numbers 
are  appreciably  decreased  only  when  large  amounts  of  rosin  are 
added.  Presence  of  rosin  greatly  increases  the  acid  number, 
which  in  pure  linseed  oil  is  usually  less  than  7.  Rosin  acids  can 
be  separated  and  determined  by  Twitchell's  method  as  described 
under  soap  analysis  beyond. 

Rosin  oil  in  linseed  oil  would  raise  the  specific  gravity  and 
greatly  lower  the  saponification  and  iodine  numbers.  Rosin  oil 
is  a  mixture  of  substances  many  of  which  are  unsaponifiable,  so 
that  its  presence  in  linseed  oil  would  increase  the  amount  of 
unsaponifiable  matter. 

Either  rosin  or  rosin  oil  can  be  detected  by  the  Liebermann- 
Storch  color  reaction  or  by  determining  the  bromine  substitution 
number. 

Liebermann-StorcTi  Reaction.  —  Shake  2  cc.  of  the  oil  with  5 
cc.  of  acetic  anhydride,  warming  gently.  Allow  to  cool,  draw 
off  the  anhydride,  and  test  by  adding  one  drop  of  sulphuric  acid 
(1:1).  A  violet  color  (not  permanent)  is  produced  in  the 
presence  of  rosin  or  rosin  oil.  Cholesterol,  which  might  be  found 
in  linseed  oil  if  fish  oil  were  present  as  an  adulterant,  gives  a 
similar  color  reaction. 

Bromine  Substitution  Number  (Mcllhiney).  —  Fatty  oils  take 
up  bromine  by  direct  addition,  little  or  no  substitution  taking 
place.  With  rosin  and  rosin  oil  much  the  greater  part  of  the 
bromine  is  taken  up  by  substitution,  a  molecule  of  hydrobromic 
acid  being  formed  for  each  molecule  of  bromine  which  disappears. 
The  hydrobromic  acid  thus  affords  a  means  of  measuring  the 
amount  of  substitution.  It  is  determined  by  adding  an  excess 
of  potassium  iodate  and  titrating  the  liberated  iodine.  The  same 
apparatus  can  be  used  as  in  the  determination  of  the  iodine 
number  and  the  manipulation  is  similar.  From  0.2  to  0.3  gram 
of  the  drying  oil  is  dissolved  in  10  cc.  of  carbon  tetrachloride 
and  20  cc.  of  a  one  third  normal  solution  of  bromine  in  carbon 
tetrachloride  is  added.  After  two  minutes  potassium  iodide  is 


208  METHODS   OF   ORGANIC   ANALYSIS 

added  and  the  excess  of  halogen  titrated  by  means  of  thiosul- 
phate  as  in  the  determination  of  the  iodine  number.  This  shows 
the  total  amount  of  bromine  which  has  disappeared.  As  soon 
as  this  titration  is  finished,  add  5  cc.  of  a  2  per  cent  solution  of 
potassium  iodate  and  titrate  the  iodine  set  free  from  the  iodate 
by  the  action  of  the  free  halogen  acids,  according  to  the  reaction : 

6  HI  +  KIO3  =  3  I2  +  KI  +  3  H2O. 

The  bromine  thus  found  is  equal  in  amount  to  that  which  has 
combined  with  the  sample  by  substitution.  For  further  details 
of  manipulation  the  reader  must  be  referred  to  the  original 
papers.1  According  to  Mcllhiney  the  bromine  substitution 
number  of  raw  or  boiled  linseed  oil  is  always  less  than  7,  while 
rosin  oil  gives  numbers  from  40  to  100  and  rosin  from  65  to  80. 

Maize  Oil 

The  presence  of  maize  oil  in  linseed  oil  lowers  the  specific 
gravity,  index  of  refraction,  iodine  number,  and  temperature 
reaction.  The  amount  which  can  be  added  without  carrying 
these  numbers  below  the  normal  limits  of  variation  will  depend 
upon  the  quality  of  the  linseed  oil  in  the  mixture.  Since  the 
maize  oil  used  as  an  adulterant  of  linseed  would  probably  not 
be  highly  refined,  the  characteristic  odor  and  taste  would  aid  in 
its  detection. 

Cottonseed  Oil 

The  "  constants  "  of  linseed  oil  would  be  lowered  by  cotton- 
seed in  the  same  way  as  by  maize  oil  and  to  a  somewhat  greater 
extent.  If  the  cottonseed  oil  had  not  been  heated,  its  presence 
would  be  detected  by  the  Halphen  test  as  described  under 
salad  oils. 

Fish  Oils 

Menhaden  and  other  fish  oils  are  often  used  as  adulterants, 
and  are  difficult  to  detect  with  certainty  since  their  "  constants  " 
are  frequently  within  the  limits  found  for  pure  linseed  oil. 
Their  presence  is  often  indicated  by  the  odor,  but  this  cannot 
be  relied  upon,  as  the  difference  in  odor  between  refined  men- 

1  Mcllhiney:  J.  Am.  Chem.  Soc.,  1894,  16,  275;  1899,  21,  1084  ;  1902,  24, 
1109.  See  also  Tolman  :  Ibid.,  1904,  26,  826. 


DRYING   OILS  209 

haden  and  low  grade  linseed  oil  is  not  so  pronounced  as  might 
be  supposed.  Lewkowitsch  recommends  the  determination  of 
the  melting  point  of  the  phytosteryl  acetate  obtained  from  the 
oil.  The  crystals  of  phytosteryl  acetate  from  pure  linseed  oil 
melt  at  128°-129°  (Bomer  and  Winter),  while  in  the  presence  of 
cholesterol  from  fish  oil  much  lower  melting  points  are  obtained. 
The  method  of  Eisenschiml  and  Copthorne,  as  adopted  by  the 
Association  of  Official  Agricultural  Chemists,  for  the  detection 
of  fish  oil  in  the  presence  of  vegetable  oils  is  as  follows : 

Dissolve  in  a  test  tube  about  6  grams  of  the  oil  in  12  cc.  of  a 
mixture  of  equal  parts  of  chloroform  and  glacial  acetic  acid. 
Add  bromine  drop  by  drop  until  a  slight  excess  is  indicated  by 
the  color,  keeping  the  solution  at  about  20°  C.  Allow  to  stand 
15  minutes  or  more  and  then  place  the  test  tube  in  boiling 
water.  If  only  vegetable  oils  are  present,  the  solution  will  be- 
come perfectly  clear,  while  fish  oils  will  remain  cloudy  or  con- 
tain a  precipitate  due  to  the  presence  of  insoluble  bromides. 

This  method  is  based  on  the  fact  that  the  bromides  of  the 
vegetable  oils,  although  they  may  be  precipitated  abundantly 
in  the  cold,  will  be  completely  soluble  in  the  mixture  of  chloro- 
form and  glacial  acetic  acid  when  heated  as  described  in  this 
test.  Boiled  linseed  oil  containing  metallic  driers  cannot  be 
tested  by  this  method  unless  the  metals  are  first  removed. 

This  method  of  Eisenschiml  and  Copthorne  is  evidently  an 
outgrowth  of  the  "  hexabromide  "  test  of  Hehner  and  Mitchell, 
the  description  of  which  follows. 

"  HEXABROMIDE  "  TEST 

Hehner  and  Mitchell a  showed  that  linseed  and  fish  oils  differ 
from  other  oils  in  yielding  considerable  quantities  of  insoluble 
bromides  when  treated  with  bromine  in  ether  solution.  They 
applied  the  test  directly  to  the  oil  as  follows : 

Dissolve  1  to  2  grams  of  oil  in  40  cc.  of  ether  acidulated  with 
glacial  acetic  acid,  cool  the  solution  to  5°,  and  add  bromine, 
drop  by  drop,  until  the  solution  is  permanently  colored  brown. 

1  Analyst,  1898,  23,  310.  Also  Mitchell  in  Allen's  Commercial  Organic 
Analysis,  4th  Ed.,  Vol.  II,  p.  28. 


210  METHODS    OF   ORGANIC    ANALYSIS 

After  standing  for  at  least  3  hours,  preferably  in  an  ice  box, 
filter  through  a  Gooch  crucible,  leaving  most  of  the  precipitate 
in  the  vessel  in  which  it  was  formed,  wash  4  times  with  ice-cold 
ether,  dry  the  precipitate  at  100°,  and  weigh. 

Linseed  oil  yields  23  to  38  per  cent  (usually  about  25  per 
cent)  of  the  insoluble  bromide,  the  amount  increasing  with  the 
iodine  number  of  the  oil.  Some  of  the  fish  oils  yield  equal  or 
greater  amounts;  but  tung,  poppy,  and  walnut  oils,  and  such 
seed  oils  as  maize  and  cottonseed,  yield  almost  none  —  according 
to  the  figures  compiled  by  Lewkowitsch  never  over  2  per  cent. 

Mitchell  regards  the  products  precipitated  in  these  tests  as 
bromides  of  mixed  glycerides  containing  one  radicle  of  linolenic 
acid  (or  an  isomeric  acid),  and  states  that  the  insoluble  bromide 
from  linseed  oil  contains  about  56  per  cent  of  bromine  and  melts 
at  143.5°  to  144°,  whereas  the  corresponding  bromides  from 
marine  animal  oils  decompose  before  melting  so  that  even  small 
amounts  of  such  oils  in  linseed  oil  can  be  detected  by  this  test. 

Lewkowitsch  recommends  that  the  test  be  applied  to  the 
mixed  fatty  acids  rather  than  to  the  oil  itself.  In  the  separa- 
tion of  the  acids  care  must  be  taken  to  avoid  oxidation  by  ex- 
posure to  the  air.  The  mixture  of  fatty  acids  from  linseed  oil 
yields  30  to  42  per  cent  of  hexabromide,  melting  to  a  clear  liquid 
at  175°  to  180°,  whereas  the  corresponding  products  from  fish, 
liver,  and  blubber  oils  do  not  melt  at  this  temperature  but  be- 
come darker  and  are  completely  blackened  at  about  200°. 
Lewkowitsch  states  that  this  test  is  capable  of  showing  the 
presence  of  10  per  cent  of  fish  oil  in  linseed  oil. 

For  further  discussion  of  this  test  see  the  papers  just  cited 
and  also  Proctor:  J.  Soc.  Chem.  Ind.,  25,  798  (1906). 

OILS  ALTERED  BY   AGE   OR  OXIDATION 

It  has  been  assumed  in  discussing  the  analytical  "  constants  " 
that  the  oils  under  examination  are  fresh  or  have  been  kept 
under  such  conditions  as  to  prevent  any  material  alteration. 
Age  alone  probably  has  no  appreciable  effect  upon  the  analyt- 
ical properties  of  commercially  pure  fatty  oils,  but  such  oils 
when  kept  for  a  long  time  in  contact  with  the  air,  for  example, 


DRYING   OILS 


211 


in  partially  filled  or  loosely  stoppered  vessels,  take  up  atmos- 
pheric oxygen  and  gradually  become  considerably  altered  in 
those  properties  which  are  commonly  regarded  as  "constants." 
This  atmospheric  oxidation  naturally  takes  place  much  more 
rapidly  with  drying  than  with  non-drying  or  semi-drying  oils, 
and  in  open  vessels  than  in  those  in  which  the  oil  is  exposed  to 
only  a  limited  amount  of  air.  It  is  probable  that  oils  which 
have  been  thus  altered  are  more  frequently  encountered  in 
analytical  work  than  has  been  supposed. 

The  influence  of  such  oxidation  upon  the  more  important 
analytical  properties  is  to  increase  the  specific  gravity,  index 
of  refraction,  and  temperature  reaction  with  sulphuric  acid, 
and  to  decrease  the  iodine  number,  the  specific  refractive 
power,1  and,  in  the  case  of  olive  oil,  the  viscosity  of  the  soap 
solution.  The  acidity  of  the  oil  may  increase  at  the  same  time, 
but  this  change  does  not  always  occur. 

The  following  results  were  obtained  upon  oils  intentionally 
exposed  to  the  air,  arid  while  large  as  compared  with  the  changes 
which  should  occur  under  ordinary  conditions  of  laboratory 
storage,  they  do  not  represent  the  maximum  change  which  may 
result  from  atmospheric  exposure. 

TABLE  19.  —  EFFECTS  OF  EXPOSURE  OF  OILS  TO  AIR 


Oil 

Iodine 
number 

Sp.  Gr. 
15.5° 
15.5° 

Index  of 
refraction 
at  15.5° 

Specific 
refractive 
power 

Specific 
tempera- 
ture 
reaction 

Olive  oil  before  exposure    .     .     . 

83.8 
77  3 

0.9165 
0  9240 

1.4712 
1  4722 

0.5141 
0  5100 

100 
127  2 

Lard  oil  before  exposure 
Same  after  exposure  

73.3 
56  2 

0.917 
0  943 

1.4697 
1.4724 

0.5122 
05010 

106 
141 

Cottonseed  oil  before  exposure    • 
Same  after  exposure  

105.2 
90.2 

0.923 
0.939 

1.4737 
1.4779 

0.5132 
0.5090 

171 

217  2 

Linseed  oil  before  exposure     .     . 

177.1 
136  9 

0.934 
0969 

1.4835 
1  4886 

0.5177 
05042 

1  Calculated  by  Landolt's  formula 


JV-1 
D 


(Ber.,  1882,  15,  1031),  in  which 


is  the  index  of  refraction  and  D  is  the  specific  gravity. 

2  These  numbers  were  determined  earlier  than  the  other  data  and  presumably 
represent  a  lesser  degree  of  change. 


212  METHODS   OF   ORGANIC   ANALYSIS 

Many  other  oils  have  been  tested  with  similar  results.  It  is 
evident  that  oils  thus  altered  are  very  likely  to  be  misjudged, 
especially  if  only  one  or  two  quantitative  determinations  are 
made.  Thus  if  only  the  specific  gravity  and  temperature  re- 
action of  the  olive  oil  had  been  determined,  the  results  would 
have  been  interpreted  as  indicating  the  presence  of  some  seed 
oil.  The  iodine  number  of  the  linseed  oil  taken  alone  would 
indicate  extensive  adulteration  with  some  oil  of  lower  drying 
power.  The  results  emphasize  the  importance  of  determining 
the  iodine  number  and  either  the  specific  gravity  or  the  index 
of  refraction  in  all  cases,  and  show  the  advantage  of  determin- 
ing the  temperature  reaction,  not  as  a  substitute  for  the  iodine 
number  but  for  comparison  with  it.  For  a  fuller  discussion  of 
this  subject  with  the  results  obtained  upon  a  number  of  other 
oils  the  reader  is  referred  to  two  papers  in  the  Journal  of  the 
American  Chemical  Society  (July,  1903,  and  May,  1905). 

As  the  result  of  this  work  it  appears  that  the  increase  in 
specific  gravity  and  the  decrease  in  iodine  number  are  almost 
exactly  proportional  to  each  other  in  non-drying  and  semi- 
drying  oils,  so  that  in  examining  an  altered  oil  belonging  to 
either  of  these  classes  the  original  iodine  number  can  be  esti- 
mated by  adding  0.8  to  the  number  found  on  the  exposed 
sample  for  each  increase  of  0.001  in  the  specific  gravity. 
When  the  original  specific  gravity  is  not  known,  the  calculation 
must  be  based  upon  the  average  specific  gravity  for  oil  of  the 
species  under  examination.  The  error  of  this  assumption  can 
hardly  be  sufficient  to  affect  the  interpretation  of  the  results. 

The  iodine  numbers  of  exposed  samples  of  linseed  and  fish  oils 
cannot  be  corrected  accurately  by  the  rule  given  for  semi-drying 
and  non-drying  oils,  the  number  thus  obtained  being  always  too 
low.  It  has  also  been  found  that  when  linseed  oil  is  thus  changed 
by  atmospheric  oxidation  the  amount  of  insoluble  bromide  which 
it  will  yield  in  the  hexabromide  test  is  greatly  reduced. 

Commercial  "blown"  oils  show  greatly  increased  specific 
gravities  and  decreased  iodine  numbers ;  the  changes  appear  to 
bear  much  the  same  relation  to  each  other  as  in  the  oils  which 
have  been  altered  by  exposure. 


DRYING   OILS  213 

"UNKNOWN"   OILS   AND   MIXTURES 

In  the  examination  of  an  unknown  oil  the  appearance,  odor, 
and  taste  should  be  compared  with  those  of  typical  oils  of 
known  purity.  In  testing  transfer  a  drop  of  the  oil  by  means 
of  a  glass  rod  to  the  back  of  the  tongue  and  note  both  the  first 
impression  and  the  after  taste.  The  odor  may  be  observed  not 
only  cold,  but  also  after  heating  a  portion  in  a  porcelain  dish  to 
140°-150°.  Also  after  cooling  sufficiently  pour  a  few  drops  of 
the  oil  into  the  palm  of  one  hand,  rub  with  the  other  and  smell 
again.  These  preliminary  superficial  observations  and  the 
determination  of  the  iodine  and  saponification  numbers  and 
either  the  specific  gravity  or  the  index  of  refraction  should 
locate  the  sample  as  one  of  a  small  group  of  oils,  after  which 
any  special  tests  available  for  the  detection  of  individual  mem- 
bers of  the  group  can  be  applied.  The  tests  described  in  this 
and  the  preceding  chapter  taken  in  connection  with  the  quanti- 
tative determinations  mentioned  enable  the  analyst,  in  the 
majority  of  cases,  to  identify  the  oil  or,  if  a  mixture,  the  prin- 
cipal constituent.  In  case  of  doubt  one  should  not  fail  to 
consult  the  larger  works,  especially  Lewkowitsch's  Oils,  Fats, 
and  Waxes. 

If  a  saponification  number  indicates  that  only  fatty  oil  is 
present,  but  the  relation  of  the  specific  gravity  to  the  iodine 
number  does  not  correspond  to  that  ordinarily  found  in  any 
pure  oil,  the  determination  of  the  specific  temperature  reaction 
and  the  acidity  will  usually  show  whether  the  discrepancy  is  to 
be  attributed  to  oxidation  or  adulteration. 

The  relative  commercial  value  will  of  course  determine  what 
oils  can  profitably  be  used  as  adulterants.  Prices  vary  greatly 
in  different  markets,  as  well  as  with  the  degree  to  which  the 
oils  are  refined,  and  are  also  likely  to  fluctuate  from  year  to 
year  so  that  no  fixed  order  of  commercial  value  can  be  given. 

The  list  of  oils  in  order  of  commercial  value  given  by  Gill 
and  by  Lewkowitsch  show  considerable  variation,  which  doubt- 
less is  due  largely  to  the  differences  between  American  and 
English  markets.  In  each  of  the  lists  the  highest-priced  oils 
are  given  first. 


214  METHODS    OF   ORGANIC    ANALYSIS 

Gill.  —  Almond,  castor,  sesame,  neatsf oot,  rape,  olive,  sperm, 
whale,  peanut  (aracbis),  linseed,  tallow,  lard,  fish,  cottonseed, 
mineral,  rosin  oil. 

Lewkowitsch.  —  Almond,  sperm,  olive,  neatsfoot,  lard,  cod 
liver,  arctic  sperm,  arachis,  poppy,  sesame,  seal,  rape,  castor, 
cottonseed,  maize,  linseed,  whale,  fish,  mineral,  rosin  oil. 

In  the  examination  of  mixtures  containing  other  than  fatty 
oils,  it  may  be  necessary  to  separate  the  mixed  fatty  acids  and 
examine  this  mixture  in  order  to  identify  the  fatty  oils  origin- 
ally present.  The  "  constants  "  of  the  mixed  fatty  acids  of 
various  oils,  as  well  as  of  many  oils  and  fats  not  mentioned  in 
this  work,  are  conveniently  tabulated  in  Lewkowitsch's  Lab- 
oratory Companion  to  the  Fat  and  Oil  Industries. 

REFERENCES 


ALLEN  :    Commercial  Organic  Analysis. 

CHURCH  :   Chemistry  of  Paints  and  Painting. 

ENNIS  :   Linseed  Oil  and  other  Seed  Oils. 

FAHRION  :   Die  Chemie  der  trocknenden  Oele. 

GILL  :    Handbook  of  Oil  Analysis. 

HOLLEY   and  LADD  :    Analysis   of   Mixed    Paints,   Color   Pigments,   and 

Varnishes. 
LEWKOWITSCH  :   Chemical  Technology  and  Analysis  of  the  Oils,  Fats,  and 

Waxes. 

SABIN  :   Technology  of  Paint  and  Yarnish. 
SCOTT  :   White  Paints  and  Painting  Materials. 
TOCH  :   Materials  for  Permanent  Painting. 
:   Technology  of  Mixed  Paints. 

II 

1891.  BALLANTYNE:    (Oxidized  Oils).     /.  Soc.  Chem.Ind.,  10,  29. 

1892.  THOMPSON  and  BALLANTYNE  :   (Same).    J.  Soc.  Chem.  2nd.,  11,  506. 

1898.  FAHRION:    (Same).     Z.  angew.  Chem.,  1898, 781. 

HEHNER  and  MITCHELL  :    ("  Hexabromide"  Test).     Analyst,  23,  310. 

1899.  GILL  and  LAMB  :   American  Linseed  Oil.     /.  Am.  Chem.  Soc.,  21,  29. 
MC!LHINEY:    (Bromine  Substitution  Number).     /.  Am.  Chem.  Soc., 

21,  1084. 

1901.  MclLHiNEY  :  Report  on  Linseed  Oil  and  its  Adulterants  to  the  New 
York  State  Commissioner  of  Agriculture.  (Reprinted  in  Sabin's 
Technology  of  Paint  and  Varnish,  Chapter  V.) 


DRYING   OILS  215 

1902.  LEWKOWITSCH  :    (Oxidized  Oils).     Analyst,  &7,  139. 
MC!LHINEY:   Further  notes  on   the   Bromine   Absorption   of   Oils. 

/.  Am.  Chem.  Soc.,  24,  1109. 

1903.  DUNLAP  and  SCHENK  :   Oxidation  of  Linseed  Oil.     J.  Am.  Chem.  Soc., 

25,  826. 
SHERMAN  and  FALK  :   Influence  of  Atmospheric  Oxidation  upon  the 

Composition   and   Analytical  Constants  of  Fatty  Oils.     J.  Am. 

Chem.  Soc.,  25,  711. 

SJOLLEMA  :   Linseed  Oil.     Z.  Ndhr.  Genussm.,  6,  631. 
UTZ  :    Poppyseed  Oil.     Chem  Ztg.,  27,  1176. 

1904.  LEWKOWITSCH:   Linseed  Oil.     Analyst,  29,  2. 

MCCANDLESS  :   Examination  of  Turpentine.     J.  Am.  Chem.  Soc.,  26, 

981. 

1904-07.  Reports  and  Discussions  on  Preservative  Coatings  for  Iron  and 
Steel.  Proc.  Am.  Soc.  Testing  Materials,  4,  137  ;  5,  79 ;  6,  63  ;  7, 
140. 

1905.  LANGMUIR  :   Determination  of  Rosin  in  Shellac.     J.  Soc.  Chem.  Ind., 

24,  12. 
McGiLL :   Examination   of  Turpentine.      Bui.  79,  Canadian  Inland 

Revenue  Laboratory. 
RABY  :   Rotatory  Power  of  Turpentine.     Ann.  de  Chim.  Anal.,   10, 

146  ;  Analyst,  30,  210. 
SHERMAN  and  FALK:   Influence  of  Atmospheric  Oxidation  upon  the 

Analytical  Constants  of  Fatty  Oils.     J.  Am.  Chem.  Soc.,  27,  605. 
THOMPSON:   Proper  Methods  in  Conducting  Painting  Tests.     Proc. 

Am.  Soc.  Testing  Materials,  5,  417. 
VALENTA  :   Examination   of   Turpentine.       Chem.   Ztg.,     29,    807 ; 

Analyst,  30,  342. 

1906.  BEADLE  and  STEVENS:    Analysis  of  Rosin  Size.     Chem.  News,  93, 

155 ;  Chem.  Eng.,  4,  263. 
BOHME  :   Detection  of  Adulterants  in  Turpentine.     Chem.  Ztg.,  30, 

631. 
GENTHER  :   Drying  Process  of  Linseed  Oil.     Z.  angew.  Chem.,  1906  ; 

Chem.  Abs.,  1,  912. 
GILL:   Determination  of  Rosin  in  Varnishes.     J.  Am.  Chem.  Soc., 23, 

1723. 
HOLLEY:    Turpentine  and  its  Substitutes.     17th  Ann.  Rpt.  North 

Dakota  Agl.  Expt.  Station. 
LEVY  :    American  Colophony.     Ber.,  39,  3043. 
PROCTOR   and  BENNETT:    Examination  of    Marine  Oils.      J.    Soc. 

Chem.  Ind.,  25,  798. 

1907.  CHEESMAN:   Priming   Coats  for  Metal   Surfaces  —  Linseed  Oil   vs. 

Paint.     Proc.  Am.  Soc.  Testing  Materials,  7,  479. 
CLOVER  :   Philippine  Wood  Oils.     Phil.  J.  Sci.,  1,  191. 


216  METHODS   OF   ORGANIC   ANALYSIS 

1907.  Committee  Report  on  Shellac  Analysis.     J.  Am.  Chem.  Soc.,  29, 1221. 
ENDEMANN  :   Testing  Shellac  for  Purity.     J.  Frank.  Inst.,  164,  285. 
HUGHES:   Deleterious    Ingredients   in    Paints     (with    Discussion). 

Proc.  Am.  Soc.  Testing  Materials,  7,  486. 
KRESS  :  Analytical  Properties  of  some  Pine  Wood  Oils.  School  of 

Mines  Quarterly,  29,  46. 
LADD:    Paint  Legislation  (with  Discussion).     Proc.  Am.  Soc.  Testing 

Materials,  7,  523. 

McGiLL  :    Examination  of  Turpentine.     J.  Soc.  Chem.  Ind.,  26,  847. 
PERRY:    Physical  Properties  of  Paint  Films.     Proc.  Am.  Soc.  Testing 

Materials,  7,  511. 
PHOKIN  :    (Oxidation  and  Polymerization  Processes  in  Drying  Oils). 

J.  Russ.  Phys.  Chem.   Soc.,  39,  307,  308 ;  Chem.  Abs.,  1,  1752, 

1775. 

RYAN  and  MARSHALL  :   Influence  of  Oxygen  and  Nitrogen,  and  San- 
light  and  Darkness  on  Olive  Oil.     Am.  J.  Pharm.,  79,  308  ;  Chem. 

Abs.,  1,  2275. 
SMITH  :   Physical  Testing  of  Oil  Varnishes  (with  Discussion).    Proc. 

Am.  Soc.  Testing  Materials,  7,  499. 
UTZ  :    Specific  Gravity  of  Linseed  Oil.     Chem.  Rev.  Fett-Harz-Ind., 

14,137;  Chem.  Abs.,  1,  2181. 

1908.  COSTE:   Examination  of  Turpentine  and  its  Substitutes.      Analyst, 

33,  219. 
FREY  :  Rapid  Determination  of  Naphtha  in   Turpentine.     J.  Am. 

Chem.  Soc.,  30,  420. 

GILL:  Oxidation  of  Olive  Oil.     J.  Am.  Chem.  Soc.,  30,  874. 
LORENTZ  :  Unsaponifiable  Matter  of  Linseed  Oil.     Chem.  Ztg.,  32, 

819. 
MCILHINEY:  Analysis  of  Oil  Varnishes.     Eng.  News,  60,  31 ;  Chem. 

Abs.,  2,  2630. 
MC!LHINEY:  Method  of  Analyzing  Shellac.     J.  Am.  Chem.  Soc.,  30, 

867. 
MARCUSSON  :  Determination  of  Benzine  in  Turpentine.     Chem.  Rev. 

Fett-Harz-Ind.,  17,  6;  Chem.  Abs.,  4,  1541. 
RICHARDSON   and   BOWEN:   Analysis  of  Turpentine  Oils.     J.  Soc. 

Chem.  Ind.,  27,  613. 
SCHULTZE:    (Nature  of  Rosin  Oil).     Ann.  Chem.,  359,  129;    Chem. 

Abs.,  2,  1715. 

1909.  Committee  Report  on  Preservative  Coatings  for  Structural  Materials. 

Proc.  Am.  Soc.  Testing  Materials,  9,  139. 
GEER  :  Analysis  of  Turpentine  by  Fractional  Distillation.  Cir.  152, 

Forest  Service,  U.  S.  Dept.  Agriculture. 
MARCUSSON  :  Turpentine  and  Its  Substitutes.  Chem.  Ztg.,  33,  966, 

978,  985;  Chem.  Abs.,  4,  1236,  1542. 


DRYING   OILS  217 

PAUL  :  Turpentine  and  Its  Adulterants.     J.  Ind.  Eng.  Chem.,  1,  27. 
WHITE:  Paints  for  Concrete  —  Their  Need  and  Requirements  (with 
Discussion).     Proc.  Am.  Soc.  Testing  Materials,  9,  526. 

1910.  AGRESTINI:    Changes  in  Olive  Oil  kept  for  Over  Two  Centuries. 

Staz.  sper.  agrar.  ital.,  43,  283 ;   Chem.  Abs.,  4,  3147. 
EIBNER  and  HUE  :  Determination  of  Benzine  in  Turpentine.     Chem. 

Ztg.,  34,  643,  657. 
EISENSCHIML  and  COPTHORNE  :  Detection  of  Fish  Oils  in  Vegetable 

Oil.     J.  Ind.  Eng.  Chem.,  2,  43. 
HEPBURN  :  Natural  Changes  Occurring  in  Fats  and  Oils.     J.  Frank. 

Inst.,  168,  365,  421 ;  169,  23. 
KREIKENBAUM  :  Analytical  Constants  of  Chinese  Wood  Oil.     J.  Ind. 

Eng.  Chem.,  2,  205. 

MORRELL  :  Testing  Turpentine.     J.  Soc.  Chem.  Ind.,  29,  241. 
SINGH  :   Analytical   Constants  of  Shellac.     J.  Soc.  Chem.  Ind.,  29, 

1435. 
YAUBEL  :  Analysis  of  Shellac.    Chem.  Ztg.,  34,  991, 1008;  Chem.  Abs., 

5,  1196. 

WALKER  :   Some  Technical  Methods  of  Testing  Miscellaneous  Sup- 
plies.    U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  109,  Revised. 

1911.  CHERCHEFFSKY:  Methods  of  Testing  Turpentine.     Matieres  grasses, 

3,  1925;  Chem.  Abs.,  5,  1677. 

COSTE  and  NASH  :  Turpentine  Substitutes.     Analyst,  36,  207. 
FORREST  :    Characteristics  of  Creosote  and  Tar  Oils  Available  for 

Wood  Preservation.     J.  Soc.  Chem.  Ind.,  30,  193. 
INGLE:    Linseed  and   Other  Oils  —  Relations  of   "Constants"  and 

Effect  of  Heat.     /.  Soc.  Chem.  Ind.,  30,  344. 
JENSEN  :   Examination  of  Linseed  Oil.     Pharm.  J.,  86,  839 ;    Chem. 

Abs.,  5,  2976. 
LANGMUIR  and  WHITE  :  The  Analysis  of  Shellac.    J.  Soc.  Chem.  Ind., 

30,  786. 

TOCH  :  Fish  Oil  as  a  Paint  Vehicle.     /.  Ind.  Eng.  Chem.,  3,  627. 
VEITCH:    Commercial  Turpentines  of  the  United  States.     J.  Ind. 

Eng.  Chem.,  3,  521. 
VEITCH  and  DONK  :   Wood  Turpentine;    Its  Production,  Refining, 

Properties,  and  Uses.     U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui. 

144. 


CHAPTER  XI 
Petroleum  and  Lubricating  Oils 

THE  petroleums  from  different  localities  vary  considerably 
both  in  the  hydrocarbons  of  which  they  are  chiefly  composed 
and  in  the  amounts  of  nitrogen,  sulphur,  and  oxygen  com- 
pounds present.  The  Appalachian  field,  which  includes  the 
oil  regions  of  Pennsylvania,  New  York,  West  Virginia,  Ken- 
tucky, and  southeastern  Ohio,  yields  petroleum  consisting 
chiefly  of  paraffin  hydrocarbons  —  of  which  all  the  members 
of  the  series  from  CH4  to  C16H34,  as  well  as  C25H52,  C27H56,  and 
C30H62,  have  been  shown  to  be  present  —  together  with  small 
amounts  of  the  olefines  and  traces  of  the  hydrocarbons  of  the 
benzene  and  napthene  series.  This  is  generally  considered 
the  best  grade  of  petroleum  produced  in  large  quantities.  It 
contains  very  little  sulphur  (between  0.05  and  0.1  per  cent) 
practically  no  asphaltic  matter  and  in  refining  gives  good 
yields  of  gasoline,  illuminating  oils,  and  paraffin  wax. 

Oils  from  the  middle  western,  Texas,  and  California  fields 
now  produced  in  much  larger  quantities  than  Pennsylvania 
petroleum,  contain  in  general  a  somewhat  lower  proportion 
of  the  paraffin  hydrocarbons,  and  larger  percentages  of  the  less 
desirable  or  undesirable  constituents. 

For  descriptions  of  the  composition  of  petroleum  oils  refer- 
ence may  be  made  to  the  works  cited  at  the  end  of  the  chapter 
and  particularly  to  a  series  of  papers  by  Mabery  in  the  "Pro- 
ceedings of  the  American  Academy  of  Arts  and  Sciences," 
Vol.  32,  and  subsequently. 

EXAMINATION   OF   CRUDE  PETROLEUM 

An  examination  of  crude  petroleum  may  consist  of  a  few 
simple  tests'  or  an  elaborate  investigation  both  by  analytical 

218 


PETROLEUM   AND    LUBRICATING   OILS  219 

methods  and  a  laboratory  imitation  of  actual  refining  processes, 
according  to  the  importance  of  the  case  and  the  judgment  and 
experience  of  the  chemist.  We  shall  consider  here  only  a  few 
factors  of  the  superficial  examination  and  fractional  distillation. 

A  sample  of  petroleum  as  received  at  the  laboratory  may 
contain  water,  either  naturally  or  because  of  having  been  put 
into  a  wet  bottle.  It  should  be  allowed  to  stand  so  that  any 
water  may  settle  out  as  completely  as  possible  before  making 
any  tests.  If  any  water  or  sediment  appear  on  standing,  de- 
cant the  oil  and  then  pass  it  through  a  dry  filter.  In  some 
cases  it  will  be  desirable  to  make  a  quantitative  determination 
of  the  moisture  and  of  the  sediment. 

Note  the  color  and  general  appearance  of  the  oil  in  a  stand- 
ard 4-oz.  sample  bottle.  With  experience  the  odor  is  also 
of  much  assistance  in  indicating  the  general  character  of  the 
sample. 

The  density  of  the  sample  is  usually  taken  with  a  delicate 
hydrometer  reading  either  in  specific  gravity  or  degrees  Baume. 
In  the  oil  trade  the  latter  is  more  common,  and  the  relation  be- 
tween specific  gravity  and  Baume  density  becomes  a  matter  of 
importance. 

In  the  past  the  different  manufacturers  of  Baume  hydrom- 
eters have  given  many  different  values  to  the  Baume  scale.1 
Recently  the  U.  S.  Bureau  of  Standards  has  defined  the  value 
of  the  Baume  scale  (for  liquids  lighter  than  water)  in  terms  of 
specific  gravity  as  follows  : 

Degrees  Baume  = —  130. 

o        •  c.  •.!_      oU     r . 

Specific  gravity  ^-^ 

The  same  value  was  adopted  by  the  Manufacturing  Chemists' 
Association  of  the  United  States  in  1903.  Hydrometers  pur- 
porting to  read  degrees  Baume  should  not  be  used  unless  they 
have  been  graduated  in  strict  conformity  with  this  scale. 

In  the  absence  of  a  sufficiently  accurate  and  delicate  hydrom- 
eter the  specific  gravity  should  be  determined  by  means  of  a 

1  Chandler  :  The  Baum^  Hydrometers.  Memoirs  of  the  National  Academy  of 
Sciences,  Vol.  Ill  (1881). 


220 


METHODS    OF   ORGANIC    ANALYSIS 


pyknometer  or  a  good  Westphal  balance.  The  results  obtained 
may  then  be  converted  to  Baume  (if  desired)  by  means  of  the 
above  equation. 

In  routine  work  it  is  customary  to  use  a  Baume  hydrometer 
with  Fahrenheit  thermometer  attached  and  add  or  subtract  0.1° 
from  the  Baume  reading  for  each  degree  Fahrenheit  below  or 

above  60°. 1  When  accuracy 
is  desired,  the  density  should 
be  observed  at  the  standard 
temperature  so  as  to  avoid 
the  necessity  for  tempera- 
ture corrections. 

As  a  rough  preliminary 
indication  of  the  purposes 
for  which  the  crude  petro- 
f  leum  may  be  useful,  it  is 
often  submitted  to  an  Engler 
distillation  test,  in  which 
100  cc.  of  the  oil  are  placed 
in  a  distilling  flask  of  the 
size  and  shape  specified  by 
Engler  (Fig.  13)  and  sub- 
mitted to  distillation,  heat- 
ing first  over  a  wire  gauze 
and  then  with  a  free  flame 
with  care  to  avoid  drafts. 
The  top  of  the  thermometer 
bulb  should  be  on  a  level 
with  the  bottom  of  the  opening  in  the  neck  of  the  flask.  Raise 
the  temperature  carefully  (especially  when  in  the  neighborhood 
of  100°,  as  here  drops  of  water  may  condense  on  the  thermometer 
bulb  and  fall  back  upon  the  hot  oil,  producing  small  explosions 
which  may  endanger  the  experiment2)  and  at  such  a  rate  as  to  dis- 
till over  as  nearly  as  possible  2*  cc.  per  minute.  When  the  ther- 

1  Better  results  are  obtained  by  the  use  of  the  correction  table  given  in 
Tagliabue's  Manual  for  Inspectors  of  Coal  Oil. 

2  Water  should  be  removed  in  advance  by  settling  as  described  above. 


FIG.  13.— Diagram  of  Engler  distillation  flask. 


PETROLEUM   AND    LUBRICATING   OILS  221 

mometer  registers  150°  C.,  remove  the  flame  and  allow  the 
temperature  to  fall  20°,  and  then  heat  again  to  150°,  cool  again, 
and  repeat  as  long  as  any  distillate  is  obtained  below  150°. 
Then  raise  the  temperature  and  collect  separately  the  second 
distillate,  which  is  usually  that  obtained  between  150°  and  200°, 
the  distillation  being  conducted  in  the  same  way  as  before. 
Similarly  collect  and  measure  the  distillates  between  200°  and 
250°,  between  250°  and  300° ;  and  finally  all  above  300°  until 
nothing  but  coke  is  left  in  the  flask. 

In  order  that  the  heavier  portions  may  not  undergo  loss  by 
hardening  in  the  cold  condenser  tube,  Low  suggests  that  the 
water  be  drained  from  the  condenser  after  the  boiling  point 
passes  200°. 

Usually  the  three  fractions  collected  between  150°  and  300° 
are  added  together  as  an  indication  of  the  "illuminating  oil," 
while  that  below  150°  is  taken  as  an  indication  of  the 
"naphtha,"  and  that  above  300°  the  "lubricating  oil,"  obtain- 
able from  the  sample. 

Grray's  method  for  oils  of  the  Pennsylvania  type  consists  in 
distilling  from  2  to  4  liters  of  the  crude  oil  by  means  of  outside 
heat  and  a  current  of  steam,  collecting  the  distillate  in  fractions 
each  of  which  is  1  per  cent  (by  volume)  of  the  sample.  The 
Baume  density  of  each  fraction  is  observed  as  the  distillation 
proceeds.  All  fractions  reading  over  57°  Baume  (lighter  than 
0.7487  specific  gravity)  are  added  together  as  "naphtha"',  those 
between  57°  and  43°  B.  (0.7487  to  0.8092  specific  gravity)  as 
"  illuminating  oils  "  ;  and  all  the  distillate  from  this  point  until 
18  per  cent  of  the  original  oil  remains  in  the  still  as  "heavy 
distillate"  the  residue  being  "steam  refined  cylinder  stock" 
unless  the  petroleum  was  of  the  asphaltic  type. 

Interpretation.  —  It  is  to  be  remembered  that  the  competent 
petroleum  expert  uses  these  fractional  distillations  only  as  one 
factor  in  the  detailed  examination  upon  which  to  base  a  final 
opinion  of  the  industrial  possibilities  of  a  crude  petroleum. 
Much  depends  upon  the  character  of  the  different  fractions  and 
their  adaptability  to  further  refining. 

Petroleums  are  sometimes  spoken  of  as  having  a  "paraffin 


222  METHODS  OF  ORGANIC  ANALYSIS 

base  "  or  an  "  asphalt  base,"  according  as  the  heavier  hydrocar- 
bons are  solid  paraffins  or  asphaltic  substances.  A  pronounced 
example  of  either  type  may  be  recognized  by  the  examination 
of  the  heavier  fractions  or  residue  obtained  in  a  fractional  dis- 
tillation, but  since  the  introduction  of  the  middle  western  oils 
and  those  from  northern  Texas,  there  is  no  sharp  dividing  line 
between  the  paraffin  and  asphaltic  base  crude  oils,  many  oils 
yielding  both  asphaltic  material  and  paraffin  wax.  When  the 
refining  process  is  a  destructive  distillation,  the  asphaltic  mate- 
rial may  be  broken  up  by  "  cracking  "  into  fuel  oil,  lubricating 
oil,  etc.,  and  the  paraffin  wax  recovered ;  but  if  the  process  is 
one  of  steam  distillation,  the  residue  being  utilized  for  cylinder 
stock,  the  asphaltic  matter  is  detrimental  to  the  quality  of  the 
cylinder  stock  obtained. 

The  question  sometimes  arises  whether  a  given  sample  is  a 
genuine  crude  oil  or  a  mixture  of  fractions  of  lesser  commercial 
or  refining  value.  In  such  cases  it  is  usual  to  distill,  collect- 
ing the  distillate  in  fractions  each  one  tenth  (by  volume)  of 
the  sample  taken,  and  determine  the  density  of  each.  A  regu- 
lar gradation  of  densities  in  the  fractions  is  an  indication  of 
genuineness  of  the  oil. 


EXAMINATION  OF  LUBRICATING  OILS 

A  thorough  examination  of  lubricating  oil  involves :  (1)  the 
determination  of  the  nature  of  the  oil  and,  if  it  is  a  mixture, 
the  proportion  of  each  constituent ;  (2)  tests  to  determine  the 
efficiency  and  safety  of  the  oil  as  a  lubricant  with  special  refer- 
ence to  the  conditions  of  temperature,  pressure,  etc.,  to  which 
it  will  be  subjected  in  use.  Among  the  more  important  prop- 
erties of  lubricating  oils  which  can  be  measured  in  the  labora- 
tory are  specific  gravity,  viscosity,  acidity,  or  alkalinity,  the 
temperature  at  which  the  oil  solidifies,  the  flashing  and  burn- 
ing points,  asphaltic  matter,  and  freedom  from  grit  or  other 
objectionable  impurities.  The  usefulness  of  other  determina- 
tions will  depend  upon  the  purposes  for  which  the  oil  is 
intended. 


PETROLEUM  AND  LUBRICATING  OILS  223 

DETERMINATION  OF  CONSTITUENTS 

Pure  fatty  and  mineral  oils  are  largely  used  as  lubricants, 
both  singly  and  mixed  with  each  other  in  all  proportions. 
Other  substances  are,  however,  often  added  to  increase  the  vis- 
cosity of  the  oil,  among  the  most  common  being  "  gelatin  oils  " 
containing  aluminium  oleate  or  other  soaps.  A  better  but  more 
expensive  means  of  increasing  viscosity  is  to  use  castor  oil  or  a 
"  blown  "  oil. 

In  beginning  the  examination  of  a  lubricating  oil,  note  care- 
fully any  color,  odor,  turbidity,  or  fluorescence  which  may  aid 
in  identifying  the  oil  or  detecting  foreign  substances.  The 
presence  of  soap  is  easily  shown  by  burning  a  weighed  portion 
of  the  oil,  as  refined  fatty  and  mineral  oils  should  not  yield 
over  0.05  per  cent  of  ash.  Rosin  oil  can  be  detected  by  the 
Liebermann-Storch  reaction. 

Qualitative  test  for  saponifiable  oil  is  conveniently  made  by 
the  Lux-Ruhemann  method  as  follows  : l  Put  3  to  4  cc.  of  the 
oil  to  be  tested  in  a  dry  test  tube,  add  a  small  piece  of  sodium 
hydroxide,  or  (better)  metallic  sodium,  and  heat  in  a  paraffin 
bath  for  15  minutes  at  230°  C.  in  the  case  of  pale-colored,  or 
250°  C.  in  the  case  of  dark-colored  or  cylinder  oils.  On  re- 
moving the  tube  from  the  bath  and  allowing  it  to  cool,  the 
presence  of  saponifiable  oil  is  indicated  by  the  partial  or  com- 
plete gelatinization  of  the  contents  of  the  tube  or  by  the  appear- 
ance of  a  soapy  froth  on  the  surface. 

After  making  these  preliminary  observations  the  saponifica- 
tion  number  should  be  determined  unless  the  sample  is  a  pure 
mineral  oil.  In  saponifying  mixtures  consisting  largely  of 
heavy  mineral  oil  there  is  difficulty  in  securing  sufficient  con- 
tact between  the  sample  and  the  alcoholic  potash  solution  even 
though  petroleum  ether  or  gasoline  be  added.  In  such  cases  a 
Soxhlet  extractor  can  be  placed  between  the  flask  and  the  re- 
flux condenser.2  The  intermittent  syphoning  of  the  condensed 
solvent  from  the  extractor  into  the  saponification  flask  mixes 

1  J.  Soc.  Chem.  Ind.,  12,  470.  Archbutt  and  Deeley  :  Lubrication  and  Lubri- 
cants, p.  209. 

2Conradson:  J.  Am.  Chem,  Soc.,  1904,  26,  672. 


224  METHODS    OF   ORGANIC   ANALYSIS 

the  contents  and  facilitates  saponification.  In  order  to  dimin- 
ish the  volume  of  solvent  required  and  the  interval  between 
stirrings,  the  body  of  the  extractor  is  partially  filled  with  glass 
beads.  Having  found  the  saponification  number  (Chapter  VIII), 
if  the  sample  appears  to  be  a  mixture,  the  proportions  of  saponi- 
fiable  and  unsaponifiable  matter  are  found  either  by  separating 
and  weighing  the  latter,  or  by  estimating  the  former  from  the 
amount  of  fatty  acids  recovered  from  the  soap  solution  after 
saponification. 

DETERMINATION  OF  UNSAPONIFIABLE  OILS 

Weigh  1  to  5  grams  of  oil  (depending  upon  the  saponification 
number  and  the  method  to  be  followed),  saponify  by  heating 
with  alcoholic  potash  on  a  water  bath,1  evaporate  off  the  alco- 
hol, and  separate  the  unsaponifiable  matter  by  one  of  the  follow- 
ing methods. 

Method  of  Immiscible  Solvents.  —  To  the  residue  from  the 
evaporation  of  alcohol  add  75  cc.  of  water,  stir  thoroughly, 
transfer  to  a  separatory  funnel,  add  about  an  equal  volume  of 
petroleum  ether  or  washed  ethyl  ether,  close  the  funnel,  shake 
vigorously,  and  allow  to  stand  over  night  or  until  the  aqueous 
and  ethereal  solutions  separate  completely.  Draw  off  the 
aqueous  layer  into  another  separatory  funnel ;  wash  it  again 
with  ether  and  the  ethereal  layer  again  with  water ;  repeat  if 
necessary.  Finally  unite  the  ether  solutions  in  a  weighed  flask,, 
distill  off  the  ether,  and  dry  the  unsaponifiable  oil  to  constant 
weight  in  a  boiling  water  oven. 

If  desired,  the  fatty  acids  can  be  recovered  from  the  aqueous 
soap  solution  by  adding  an  excess  of  mineral  acid  and  shaking 
with  ether  or  by  separating  the  fatty  acids  as  in  soap  analysis. 

The  principal  objection  to  the  separation  by  immiscible  sol- 
vents is  that  emulsions  frequently  form  in  the  separating  funnel 
which  remain  even  on  standing  for  a  day  or  more.  The  addi- 
tion of  1  to  2  cc.  of  alcohol  often  helps  to  break  the  emulsion, 

1  See  also  the  method  involving  cold  saponification  given  by  Fahrion  :  Z. 
angew.  Chem.,  1898,  267. 


PETROLEUM   AND   LUBRICATING   OILS  225 

but  if  more  alcohol  is  added  it  tends  to  carry  soap  into  the 
ether  layer.  The  separation  of  the  solvents  is  also  facilitated 
by  chilling  the  funnel  and  twirling  it  gently,  or,  if  the  apparatus 
is  available,  by  whirling  in  a  centrifuge.  Petroleum  ether  dis- 
solves less  soap  than  ethyl  ether  and  gives  less  troublesome 
emulsions,  but  does  not  always  extract  the  unsaponifiable  mat- 
ter completely. 

Extraction  of  the  Dry  Soap.  —  To  avoid  the  difficulties  just 
noted  the  following  modification  of  the  method  recommended 
by  A.  C.  Wright1  may  be  used:  Saponify  2  to  4  grams  of 
oil,  using  2  grams  of  caustic  potash ;  after  evaporating  off  the 
alcohol,  add  3  grams  of  sodium  bicarbonate  and  10  cc.  of  pure 
methyl  alcohol,  stir  well  and  evaporate,  add  5  cc.  more  of 
methyl  alcohol  and  10  grams  of  precipitated  chalk,  mix  well, 
dry  on  a  water  bath  and  then  for  a  few  minutes  at  110°. 
Transfer  the  thoroughly  dried  mixture  quickly  to  a  Soxhlet 
extractor  and  extract  the  unsaponifiable  matter  with  petroleum 
ether.  Dry  the  extract  to  constant  weight  in  a  boiling  water 
oven  and  weigh. 

The  mixture  of  calcium  carbonate  and  soap  from  which  the 
unsaponifiable  matter  has  been  extracted  can  be  treated  with 
hydrochloric  acid  to  dissolve  the  carbonate  and  liberate  the 
fatty  acids,  which  can  then  be  separated  and  examined  further. 

ESTIMATION  AND  IDENTIFICATION  OF  FATTY  OILS 

From  the  weight  of  fatty  acid  recovered  as  described  above, 
the  percentage  of  fatty  oil  can  be  calculated  on  the  assumption 
that  the  oil  yields  95  per  cent  of  insoluble  fatty  acids.  The 
result  thus  found  serves  as  a  check  upon  the  determination  of 
unsaponifiable  oil. 

If  only  fatty  and  mineral  oils  are  present  and  the  percentage 
of  the  former  is  small,  it  can  be  estimated  with  sufficient  ac- 
curacy for  most  purposes  from  the  saponification  number,  since 
the  fatty  oils  which  are  likely  to  be  present  in  mixed  lubricants 
do  not  vary  greatly  in  their  saponification  numbers.  See  table 

1  Analysis  of  Oils  and  Allied  Substances,  p.  111. 


226  METHODS    OF   ORGANIC    ANALYSIS 

at  end  of  Chapter  VIII.  If  the  fatty  oil  is  identified,  the  aver- 
age number  for  that  species  of  oil  should  be  used  in  estimating 
the  percentage. 

If  the  lubricant  consists  entirely  of  fatty  oil  with  a  known 
small  amount  of  inert  unsaponifiable  matter,  the  usual  methods 
for  the  identification  of  fatty  oils  can  be  employed.  Otherwise 
the  identification  is  based  upon  the  examination  of  the  separated 
fatty  acids. 

VISCOSITY 

Apparatus  and  Methods 

The  viscosity  of  an  oil  can  be  determined  either  by  measur- 
ing the  resistance  which  it  offers  to  the  movement  of  a  sub- 
merged solid,  or  by  observing  the  rate  at  which  it  flows  through 
an  aperture  under  given  conditions  of  temperature  and  pressure. 
Torsion  viscosimeters,  such  as  that  of  Doolittle,  depend  upon 
the  first  principle ;  but  those  depending  upon  the  measurement 
of  the  rate  of  flow  are  much  more  generally  used.  Viscosime- 
ters of  this  kind  are  made  in  a  great  variety  of  forms,  for  de- 
scriptions of  which  the  reference  books  at  the  end  of  the  chapter 
can  be  consulted.  Among  the  viscosimeters  most  commonly 
used  are  those  of  Engler,  Redwood,  and  Saybolt. 

The  Engler  viscosimeter  is  probably  more  widely  used  the 
world  over  than  any  other  form.  It  has  long  been  regarded  as 
the  standard  instrument  in  Germany,  and  has  now  been  adopted 
in  the  United  States  government  specifications,  and  by  the 
American  Society  for  Testing  Materials. 

It  consists  of  a  cylindrical  reservoir  with  concave  bottom,  in 
the  center  of  which  is  a  capillary  outlet.  The  reservoir  is  of 
brass,  sometimes  gold-lined,  is  surrounded  by  a  water  or  oil 
jacket,  and  provided  with  a  cover  through  which  passes  a  ther- 
mometer for  taking  the  temperature  of  the  oil  and  a  plug  which 
closes  the  outlet.  In  testing  an  oil,  240  cc.  are  poured  into  the 
reservoir,  and  should  fill  it  to  the  points  of  the  studs  which 
serve  to  indicate  the  correct  leveling  of  the  apparatus.  The 
reservoir  is  then  covered,  the  temperature  regulated,  and  finally 
the  plug  is  withdrawn,  and  the  time  required  for  the  outflow  of 


PETROLEUM   AND    LUBRICATING   OILS 


227 


200  cc.  is  carefully  noted.  The  time  in  seconds  required  by 
the  oil  divided  by  that  required  by  water  at  20°  C.  is  taken  as 
the  Engler  viscosity  number.  Unless  the  oil  is  too  viscous,  or 
for  some  other  reason  a  higher  temperature  is  desired,  it  should 
be  tested  at  20°  C.  In  any  case  the  temperature  should  be 
stated  in  reporting  results.  The  instrument  must  be  very 
carefully  cleaned  and  dried  before  and  after  using,  as  any  trace 
of  oil  around  the  outlet 
will  interfere  with  the 
flow  of  water,  and  vice 
versa.  Serious  errors 
may  also  be  caused  by 
dust,  grit,  or  scratches 
about  the  outlet.  The 
time  of  flow  of  200  cc. 
water  from  the  Engler 
viscosimeter  should  be 
from  51  to  53  seconds. 
The  "  normal  apparatus  " 
is  made  according  to 
strict  specifications,  and 
is  carefully  standardized 
by  the  German  officials. 
Figure  14  shows  a  sec- 
tion of  the  apparatus  in 
which  A  represents  the 
oil  cylinder,  B  the  outer 
bath  or  jacket,  C  the  flask 
marked  at  200  and  240  cc.  which  serves  both  to  measure  the 
oil  for  the  test  and  to  receive  it  as  it  flows  from  the  capil- 
lary, b  the  plug,  t  the  thermometer,  D  the  tripod  support,  and 
d  the  ring  burner,  which  is  used  only  when  making  tests  at 
temperatures  above  that  of  the  room.  The  correct  dimensions 
of  the  apparatus  are  also  indicated  on  this  cut. 

Redwood's  viscosimeter,  which  has  been  largely  used  in  Great 
Britain,  consists  of  a  cylinder  about  4.7  cm.  in  diameter  and 
8.7  cm.  high,  having  in  the  center  of  the  bottom  a  cup-shaped 


FIG.  14.  —  Diagram  of  Engler  viscosimeter. 

From  Lewkowitsch's  Oils,  Fats,  and  Waxes. 

(Macmillan  and  Co.) 


228  METHODS    OF   ORGANIC    ANALYSIS 

agate  jet  which  can  be  closed  by  means  of  a  spherical  plug. 
Inside  the  cylinder  is  a  small  fixed  bracket  of  thick  bent  wire 
with  an  upturned  point  to  indicate  the  height  to  which  the  oil 
should  extend  at  the  beginning  of  the  test.  The  apparatus  is 
jacketed  and  provided  with  a  closed  side  tube  and  a  revolving 
stirrer  so  that  determinations  can  be  made  at  high  temperatures 
if  desired.  The  apparatus  is  intended  to  deliver  50  cc.  of 
water  at  15.5°  in  25.5  seconds,  but  as  the  rate  of  flow  is  in- 
fluenced by  many  conditions,  it  must  be  determined  by  each 
observer  for  his  own  apparatus  and  method  of  working. 

To  use  the  apparatus  at  room  temperature  place  it  on  a  level 
support,  insert  the  plug,  and  fill  with  the  liquid  to  be  tested 
until  the  surface  comes  exactly  to  the  upturned  point  already 
mentioned.  Place  beneath  the  outlet  a  narrow-necked  flask 
graduated  at  50  cc.,  open  the  jet  by  lifting  the  ball  valve,  and 
observe  the  time  required  for  50  cc.  to  flow  into  the  receiving 
flask.  Whatever  method  is  adopted  for  expressing  the  results, 
the  report  should  always  show  the  actual  time  of  flow  for  the 
oil  and  for  water  and  the  temperature  at  which  the  test  was 
made.  The  same  precautions  should  be  taken  with  this  as  with 
the  Engler  apparatus. 

For  a  description  of  Saybolt's  viscosimeter  consult  Gill's  Oil 
Analysis. 

Significance  of  Results 

Since  the  object  of  lubricating  with  oil  is  to  separate  the 
moving  surfaces  by  a  fluid  layer,  it  is  important  that  the  oil 
have  sufficient  viscosity  or  "  body "  to  stay  in  place  and  keep 
the  moving  surfaces  apart  under  the  maximum  pressure  to 
which  they  will  be  subjected.  The  greater  the  pressure  the 
more  viscous  the  oil  should  be,  but  any  viscosity  beyond  that 
which  is  necessary  to  keep  the  oil  in  place  means  an  increase 
of  fluid  friction  and  consequent  loss  of  power.  The  viscosity 
of  the  oil  is,  therefore,  the  most  direct  indication  of  its  suita- 
bility as  a  lubricant  under  given  conditions.  For  several  rea- 
sons, however,  the  viscosity  alone  is  not  a  conclusive  measure 
of  the  lubricating  power.  The  adhesion  to  solid  surfaces  which 


PETROLEUM   AND   LUBRICATING   OILS  229 

prevents  the  displacement  of  the  oil  by  pressure  is  not  always 
directly  proportional  to  the  true  viscosity  or  internal  friction. 
Oils  vary  greatly  in  the  rate  of  change  of  viscosity  with  in- 
creasing temperature  and  pressure.  The  viscosity  as  measured 
by  the  rate  of  flow  depends  not  only  upon  the  internal  friction 
of  the  oil,  but  also  to  some  extent  upon  its  adhesion  to  the  sides 
of  the  outlet  and  upon  the  specific  gravity.  Hence,  it  is  not  to 
be  assumed  that  any  two  oils  having  the  same  viscosity  can  be 
•used  interchangeably  as  lubricants.  In  order  to  duplicate  an 
oil  which  has  been  found  satisfactory  in  use,  the  kind  of  oil,  the 
specific  gravity,  and  the  viscosity  at  least,  should  be  specified. 
Viscosity  is  especially  important  in  dealing  with  mineral  oils 
because  of  the  ease  with  which  they  can  be  varied  in  this  re- 
spect, while  any  particular  kind  of  fatty 'oil  varies  only  within 
comparatively  narrow  limits.  For  a  full  theoretical  discussion 
of  viscosity  and  lubrication,  the  work  of  Archbutt  and  Deeley 
should  be  consulted.  The  reader  must  also  be  referred  to 
this  and  other  books  and  articles  given  below,  for  discussion  of 
the  many  practical  considerations  affecting  the  selection  of 
lubricating  oils. 

Mixed  oils  do  not  always  show  the  viscosities  which  would 
be  expected  from  the  proportions  and  viscosities  of  the  con- 
stituents of  the  mixture.  In  other  words,  viscosity  is  not  an 
additive  property  in  oil  mixtures.  In  mixtures  of  oils  whose 
viscosities  are  similar,  the  discrepancy  between  the  estimated 
and  observed  viscosity  of  the  mixture  may  not  be  apparent, 
but  where  oils  of  widely  different  viscosities  are  mixed,  the 
discrepancy  may  be  considerable. 

Sherman,  Gray,  and  Hammerschlag 1  in  a  series  of  experiments 
with  nine  sets  of  mixtures  found  that  the  observed  viscosity  of 
the  mixture  was  lower  than  the  calculated  value  whether  the 
mixture  was  that  of  two  mineral  oils,  a  mineral  and  a  fatty  oil, 
or  a  mineral  oil  and  sperm  oil;  and  in  general,  the  greater  the 
difference  in  viscosities  between  the  oils  mixed,  the  greater  was 
the  difference  between  the  calculated  and  observed  viscosity 
numbers  of  the  mixtures.  In  a  typical  case  a  series  was  pre- 
ij;  Ind.Eng,  Chem.,  1,  13. 


230 


METHODS  OF  ORGANIC  ANALYSIS 


pared,  consisting  of  mixtures  in  tenths  by  weight  of  a  high 
viscosity  lubricating  oil  ("  H  ")  and  a  low  viscosity  lubricating 
oil  ("L"),  both  made  from  Pennsylvania  petroleum.  The  pure 
oils  and  mixtures  tested  as  follows: 


ENGLER  VISCOSITIES  AT  20°  C. 


Calculated 

Found 

Difference 

Hisrh  viscosity  oil  (;'H") 

25.56 

90  per  cent  "  H,"  10  per  cent  "  L  "  •   .     .     .     . 

23.41 

20.04 

3.37 

80  per  cent  "  H,"  20  per  cent  "  L  "      .... 

21.27 

16.25 

5.02 

70  per  cent  "  H,"  30  per  cent  "  L  "      .... 

19.12 

13.37 

5.75 

60  per  cent  "  H,"  40  per  cent  "  L  "      .... 

16.97 

10.90 

6.07 

50  per  cent  "  H,"  50  per  cent  "  L  "      .... 

14.83 

9.04 

5.79 

40  per  cent  "  H,"  60  per  cent  "  L  "      .... 

12.68 

7.69 

4.99 

30  per  cent  "  H,"  70  per  cent  "  L  "      .... 

10.54 

6.38 

4.16 

20  per  cent  "  H,"  80  per  cent  "  L  "      .... 

8.39 

5.52 

2.87 

10  per  cent  "  H,"  90  per  cent  "  L  "      .... 

6.24 

4.67 

1.57 

Low  viscosity  oil  C"  L  ") 

4.10 

In  every  case  the  viscosity  of  the  mixture  was  less  than  the 
value  obtained  by  calculation  from  the  percentages  and  viscosi- 
ties of  the  constituents.  The  difference  between  the  calculated 


.,3 

i 


20$       3o#       405*       sojj       6056       70:* 

Percentage  of  lower  viscosity  oil. 
FIG.  15.  —  Viscosities  of  oil  mixtures. 


9036 


PETROLEUM   AND    LUBRICATING    OILS  231 

and  determined  values  increased  with  the  increasing  propor- 
tions of  light  oil  in  the  mixture  up  to  40  per  cent  of  light  and 
60  per  cent  of  heavy  oil  ;  with  further  increments  of  the  light 
oil,  the  difference  gradually  decreased.  The  determined  vis- 
cosities are  plotted  in  Fig.  15  (the  viscosities  as  ordinates,  the 
percentages  of  "  L  "  as  abscissae),  and  will  be  seen  to  form  a 
very  regular  curve,  dropping  away  from  the  calculated  values 
somewhat  more  abruptly  when  the  light  oil  is  added  to  the 
heavy  than  when  the  heavy  oil  is  added  to  light. 

Subsequently  Kessler  and  Mathiason 1  obtained  similar  results. 

ACIDITY 

Weigh  accurately  5  to  10  grams  of  oil  in  a  250-cc.  Erlen- 
meyer  flask,  add  50  cc.  of  neutralized  85  per  cent  alcohol  con- 
taining phenolphthalein  as  indicator,  and  titrate  with  standard 
sodium  or  potassium  hydroxide,  shaking  vigorously  after  each 
addition  until  a  permanent  pink  color  is  obtained.  It  is  often 
necessary  to  allow  the  flask  to  stand  after  shaking  until  the  oil 
separates  from  the  alcohol  solution  before  observing  the  color 
of  the  latter.2 

To  test  for  free  mineral  acid,  shake  10-15  grams  of  oil  with 
100  cc.  of  warm  water  in  a  separatory  funnel,  allow  to  separate, 
draw  off  the  water,  filter  through  wet  paper,  cool,  and  add 
methyl  orange.  If  mineral  acid  is  found,  shake  the  oil  remain- 
ing in  the  funnel  repeatedly  with  small  portions  of  hot  water 
until  all  mineral  acid  is  extracted,  filter  as  before,  add  the  filtrate 
to  the  first  portion  containing  methyl  orange,  and  titrate  very 
carefully  with  standard  alkali.  Concentrate  the  neutralized 
solution,  test  qualitatively  to  determine  the  nature  of  the  min- 
eral acid,  and  calculate  the  percentage.  If  the  identification  of 
the  mineral  acid  is  prevented  by  the  presence  of  salts,  calculate 
the  mineral  acidity  as  due  to  sulphuric  acid.  The  acidity  due 

1  J.  Ind.  Eng.  Chem.,  3,  66. 

2  For  determining  acidity  in  very  dark  colored  fats  the  use  as  indicator  of 
10  cc.  of  a  2  per  cent  solution  of  "  Alkali  Blue  II  OLA  "   (Meister,  Lucius  and 
Brunig)  in  99  per  cent  alcohol  has  been  recommended  by  Freundlich  :   Oesterr. 
Chem.  Ztg.,  1901,  4,  441  ;  Z.  Nahr.  Genussm.,  1902,  5,  460. 


232  METHODS   OF   ORGANIC   ANALYSIS 

to  organic  acids,  or  the  total  acidity  if  only  this  is  determined, 
is  usually  calculated  as  percentage  of  oleic  acid.  As  much  as 
15  per  cent  of  free  oleic  acid  is  sometimes  allowed  in  lubricating 
oils.  The  best  grades  of  lard  oil  do  not  contain  over  1.5  per 
cent.  Free  mineral  acids  should  be  absent. 


COLD  TEST  AND   CHILLING  POINT  OR  CLOUD  TEST 

The  "cold  test"  indicates  the  temperature  at  which  the 
sample  just  ceases  (or  just  begins)  to  flow  ;  the  "chilling  point" 
that  at  which  the  oil  begins  to  become  turbid  or  to  show  flocks 
or  scales  of  solid.  In  either  case  the  temperature  required  will 
be  influenced  by  details  of  manipulation,  so  that  an  arbitrary 
method  must  be  followed  to  obtain  strictly  comparable  results. 
The  directions  below  follow  the  procedure  recommended  by 
Gray. 

Gold  Test.  —  Pour  about  25  cc.  of  oil  into  an  ordinary  bottle 
of  about  100  cc.  capacity  and  insert  a  stopper  carrying  a  ther- 
mometer the  bulb  of  which  reaches  just  below  the  surface  of 
the  oil,  cool  the  sample  slowly  to  50°  F.  and  then  place  in  a 
freezing  mixture  of  ice  and  salt.  As  the  temperature  falls 
every  few  degrees  remove  and  tilt  the  bottle  until  the  tempera- 
ture is  found  where  the  oil  just  ceases  to  flow.  This  is  called  the 
"cold  test"  or  "setting  point."  Cylinder  and  black  oils  which 
have  not  been  treated  either  by  acid  or  by  filtration  through 
fuller's  earth  may  show  abnormally  low  and  irregular  cold  tests. 

With  oils  having  cold  tests  higher  than  45°  F.,  it  is  custom- 
ary to  reverse  the  process,  freezing  the  sample  first  and  allowing 
it  then  to  warm  slowly  until  it  just  flows. 

Chilling  Point.  —  Usually  it  is  only  necessary  to  know  whether 
the  oil  remains  clear  for  a  given  number  of  minutes  at  a  given 
temperature.  Use  the  same  bottle,  amount  of  sample,  and  ther- 
mometer as  for  the  cold  test.  Expose  the  liquid  to  cold,  stirring 
with  the  thermometer,  and  hold  at  the  required  temperature  for 
the  specified  time  (usually  ten  minutes).  If  the  oil  remains 
transparent  and  free  from  flocks  or  scales,  it  meets  the  require- 
ment as  to  chilling  test. 


PETROLEUM   AND   LUBRICATING   OILS  233 

If  it  is  required  to  find  the  chilling  point,  the  procedure  is 
similar ;  but  the  liquid  after  remaining  clear  as  described  is  ex- 
posed to  a  temperature  3°  lower,  allowed  to  stand  with  constant 
watching  arid  occasional  stirring  with  the  thermometer  until  the 
oil  is  as  cold  as  the  bath,  repeat  this  cooling  until  opacity  or 
flocks  or  scales  begin  to  show.  The  reading  of  the  thermometer 
when  this  occurs  shows  the  "chilling  point"  or  "cloud  test." 

For  further  information  on  the  cold  test  or  setting  point  see 
the  works  of  Archbutt  and  Deeley,  Gill,  Holde,  Lewkowitsch, 
and  Stillman. 

FLASHING  AND  BURNING  POINTS 

For  the  most  accurate  results  closed  testers  such  as  are  used 
in  examining  illuminating  oils  should  be  employed.  Among 
these  the  Perisky-Martens  apparatus  is  perhaps  the  best  for  this 
purpose.  An  illustrated  description  of  this  apparatus  will  be 
found  in  Lewkowitsch's  Oils,  Fats,  and  Waxes,  4th  Ed.  Vol. 
III.  pp.  58-60. 

For  routine  work  in  this  country  the  "  Cleveland  Cup"  tester 
is  often  used.  This  is  a  shallow,  cylindrical,  jacketed  cup,  open 
to  the  air  and  heated  by  a  Bunseii  flame.1  The  test  cup  is  filled 
to  about  0.5  cm.  from  the  top  and  the  thermometer  is  suspended 
in  such  a  position  that  the  bulb  is  entirely  immersed  in  the  oil 
at  the  center  of  the  dish  without  touching  the  bottom.  Heat 
by  means  of  a  Bunsen  burner,  and  as  the  flashing  point  is 
approached,  test  at  each  second  or  third  degree  by  slowly  pass- 
ing the  test  flame  across  the  dish  horizontally  about  0.5  cm. 
above  the  level  of  the  oil  and  directly  in  front  of  the  thermom- 
eter. Record  the  temperature  at  which  the  first  flash  is  seen 
as  the  flashing  point.  Continue  heating  and  testing  in  the  same 
way  until  the  liquid  takes  fire  ;  note  this  temperature  as  the 
burning  point.  Remove  the  thermometer  and  blow  out  the 
flame  or  smother  it  by  sliding  a  watch  glass  over  the  dish. 

Notes.  —  The  flashing  and  burning  points  must  be  determined 
in  a  place  free  from  drafts.  The  heating  should  be  so  regulated 

1  In  the  absence  of  either  of  these  forms  of  apparatus  a  porcelain  dish,  4  cm. 
deep  and  4  cm.  in  diameter,  set  deep  in  a  sand  bath,  is  sometimes  used. 


234  METHODS   OF   ORGANIC   ANALYSIS 

that  on  approaching  the  flashing  or  burning  point  the  rate  of 
rise  of  temperature  of  the  oil  is  not  greater  than  6°  C.  per  min- 
ute. Tests  may  then  be  made  every  half  minute  using  a  test 
flame  not  over  5  mm.  long.  The  test  flame  may  be  obtained 
from  a  narrow  glass  jet  (similar  to  that  used  on  a  wash  bottle) 
connected  with  the  ordinary  gas  tubing,  the  flow  of  gas  being 
regulated  to  give  a  flame  of  the  size  desired.  Any  variation 
of  the  conditions,  either  in  size  and  form  of  dish,  the  rate  of 
heating  and  testing,  or  the  manner  of  applying  the  test  flame 
may  cause  an  appreciable  discrepancy  in  the  result. 

In  oil  mixtures  the  flashing  and  burning  points  are  not  addi- 
tive properties  and  cannot  be  predicted  by  simple  interpolation. 
Sherman,  Gray,  and  Hammerschlag  1  found  that  in  mixtures  of 
mineral  oils  of  different  flashing  and  burning  points,  and  of 
mineral  oil  with  a  fatty  oil,  or  sperm  oil,  the  flashing  and  burn- 
ing points  found  were  invariably  lower  than  the  figures  which 
would  be  found  by  simple  interpolation  from  the  known  prop- 
erties and  proportions  of  the  oils  in  the  mixture.  It  was  also 
observed  that  when  a  high-test  and  a  low-test  oil  were  mixed 
in  different  proportions  the  discrepancy  was  greater  in  mixtures 
containing  a  high  proportion  of  the  high-test  oil  ;  or,  differently 
stated,  the  flashing  and  burning  points  were  lowered  by  the 
presence  of  25  per  cent  of  the  low-test  oil  to  a  greater  extent 
than  they  were  raised  by  the  presence  of  25  per  cent  of  the 
high-test  oil. 

Kessler  and  Mathiason  2  have  also  shown  that  the  flashing  and 
burning  points  are  not  additive  properties  in  oil  mixtures. 

ADDITIONAL  DETERMINATIONS 

Additional  tests  and  determinations  are  frequently  required 
to  show  the  suitability  of  the  lubricant  for  the  particular  use 
intended.  Friction  tests  on  oil  testing  machines  especially 
designed  for  the  work  are  sometimes  of  great  importance.  A 
full  discussion  of  such  mechanical  methods  of  testing  will  be 
found  in  Archbutt  and  Deeley's  Lubrication  and  Lubricants. 

1  J.  Ind.  Eng.  Chem.,  1,  13.  2  J.  Ind.  Eng.  Chem.,  3,  66. 


PETROLEUM   AND    LUBRICATING   OILS  235 

Loss  by  evaporation  and  tendency  to  "gum"  are  tested  by 
heating  a  small  amount  of  oil  on  a  watch  glass  for  several  hours 
at  the  highest  temperature  to  which  it  is  likely  to  be  subjected 
in  use.  The  oil  must  not  become  sticky  and  the  loss  of  weight 
should  usually  be  less  than  1  per  cent.  Suspended  matter 
which  may  be  invisible  in  a  dark  oil  is  detected  by  diluting  the 
sample  with  gasoline  or  petroleum  ether.  Antifluorescents, 
added  to  destroy  the  fluorescence  or  u  bloom  "  of  mineral  oils, 
can  often  be  detected  by  boiling  1  cc.  of  the  oil  with  3  cc.  of  a 
10  per  cent  solution  of  potassium  hydroxide  in  alcohol.  A  red 
color  indicates  nitronaphthalene  or  nitrobenzene,  which  are 
the  principal  antifluorescents  used.  According  to  Holde,1 
asphaltic  matter  can  be  approximately  determined  as  follows: 
Dissolve  5  grams  in  125  cc.  of  ether  at  15°,  add,  drop  by  drop 
with  constant  shaking,  62.5  cc.  of  96  per  cent  alcohol  ;  after 
standing  5  hours  at  15°,  filter,  wash  with  a  mixture  of  alcohol 
and  ether  (1 :  2  by  volume)  until  nothing  more  than  traces  of 
pitchlike  substance  is  removed.  Dissolve  the  residue  in  ben- 
zol, evaporate,  dry  one  half  hour  at  105,°  and  weigh. 

For  other  tests  and  determinations  and  fuller  discussions  of 
most  of  those  here  given  the  reader  is.  referred  to  the  works 
given  for  reference  below. 

EXAMINATION  OF  LUBRICATING  GREASES 

Lubricating  greases  are  usually  mixtures  of  soaps  with  fats, 
hydrocarbons,  rosin,  or  tar,  containing  water  and  sometimes 
large  amounts  of  mineral  matter.  On  melting,  the  grease  often 
separates  into  a  soap  solution  and  an  oily  layer.  The  soaps 
used  in  making  such  lubricants  may  contain  salts  of  sodium, 
potassium,  calcium,  or  heavy  metals  with  either  fatty  or  resin 
acids.  Some  greases  consisting  essentially  of  fats  and  hydro- 
carbons melt  at  temperatures  to  which  they  are  subjected  in 
use  and  may  therefore  be  examined  in  the  melted  state  by  the 
methods  used  for  lubricating  oils.  For  most  greases,  however, 
it  is  necessary  to  adapt  the  analytical  method  to  the  nature  of 

lMitt.  Kgl.  Techn.  Versuchsanstalt,  Berlin,  1902,  20,  253;  Z.  Nahr. 
Genussm.,  1903,  6,  855. 


236  METHODS    OF   OEGANIC    ANALYSIS 

the  mixture  to  be  examined  in  each  case,  since  the  composition 
of  these  greases  is  too  variable  to  allow  the  use  of  any  fixed 
system  of  examination.  It  may  often  be  necessary  to  resort  to 
a  combination  of  the  methods  used  in  the  analysis  of  soaps,  fats, 
and  lubricating  oils.  For  detailed  information  on  the  composi- 
tion and  testing  of  lubricating  greases,  see  the  reference  books 
cited  below  and  a  review  by  Conradson:  J.  Am.  Ohem.  >SW., 
1904,  26,  705-712. 

REFERENCES 


ALDER- WRIGHT  and  MITCHELL  :   Oils,  Fats,  Butters,  and  Waxes. 

ALLEN  :   Commercial  Organic  Analysis. 

ARCHBUTT  and  DEELEY  :   Lubrication  and  Lubricants. 

DAVIS  :    Friction  and  Lubrication. 

GILL  :    Short  Handbook  of  Oil  Analysis. 

HOLDE  :    Untersuchung  der  Schmiermittel. 

LEWKOWITSCH  :   Oils,  Fats,  and  Waxes. 

LUNGE  :   Chemisch-technische  Untersuchungsmethoden. 

POST  :    Chemisch-technisehe  Analyse. 

RAKUSIN  :   Die  Untersuchung  des  Erdoles  und  seine  Producte. 

REDWOOD  :   Petroleum  and  its  Products. 

STILLMAN  :   Engineering  Chemistry. 

UBBELOHDE  :    Handbuch  der  Chemie  und  Technologic  der  Oele  und  Fette. 

II 

1904.  EGER  :    Testing  of  Mineral  Lubricating  Oils.     Z.  angew  Chem.,  1904, 

1577. 

1905.  RICHARDSON  and  HANSON  :   Valuation  of  Lubricants  with  Special 

Reference  to  Cylinder  Oils.     J.  Soc.  Chem.  Ind.,  24,.  315. 

1906.  GILL  :    Apparatus  for  Testing  Liability  of  Oils  to  produce  Spontane- 

ous Combustion.     J.  Soc.  Chem.  Ind.,  26,  185. 
Ross  and  LEATHER  :   Valuation  of  Oils  for  Gas-making.     Analyst, 

31,  284. 
TOLMAN  :   Cooperative  Work  on  the  Cloud  and  Cold  Test  for  1906. 

U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  105,  p.  29. 
VALENTA  :   Determination  of  Coal  Tar  Oils  in  Mineral  and  Rosin 

Oils.     Chem.  Ztg.,  30,  266. 

1907.  ACHESON  :    A  New  Lubricant.     Defloculated  Graphite.     Eng.  News, 

53,  127. 


PETROLEUM   AND    LUBRICATING   OILS  237 

CHARITSCHKOW:   Influence  of  Water  on  Flash-Point  and  Viscosity. 

Chem.  Rev.  Fett-  Harz-Ind.,  14,  225  :  Chem.  Abs.,  2,  176. 
KISSLING:    Constants  in  Mineral   Oil  Analysis.      Chem.  Ztg.,  31r 

328. 
Physikalisch-technische  Reichsanstalt.    Calibration  of  Engler  Visco- 

simeters.     Z.  angew.  Chem.,  20,  832 ;  Z.  offenil.  Chem.,  13,  204. 
SCHLICHT  and  HALPHEN:   Determination  of  Unsaponifiable  Matter. 

Chem.  Ztg.,  31,  279. 

SCHREIBER  :   Determination  of  Saponification  Number  in  Lubricat- 
ing Oils.     /.  Am.  Chem.  Soc.,  29,  74. 
STOLZENBURG  :   Technical  Examination  of  Lubricating  Oils.     Chem. 

Rev.  Fett-  Harz-Ind.,  14,  239,  274 ;    Chem.  Abs.,  2,  456. 
UBBELOHDE:    Improvements  in  Engler  Viscosimeter.     Chem.  Ztg., 

31,  38. 

1908.  HOLDE  :    (Physical  Behavior  of  Lubricants).    Z.  angew.  Chem.,  1908, 

31,  2138. 

HOLDE  and  EICKMANN  :  Resinous  Products  in  Mineral  Oils.  Pe- 
troleum, 1908,  3,  1077;  Chem.  Abs.,  2,  1341. 

KISSLING  :  New  Constants  in  Mineral  Lubricating  Oil  Analysis. 
Chem.  Ztg.,  32,  938. 

LECOCQ  :  Determination  of  Asphalt  in  Mineral  Oils.  Bull.  Soc. 
Chim.  Belg.,  22,  81 ;  Chem.  Abs.,  2,  1880. 

MABERY  and  MATHEWS  :  Viscocity  and  Lubrication.  J.  Am.  Chem. 
Soc.,  30,  992. 

Review  on  Lubrication.     Engineering,  85,  86;  Chem.  Abs.,  2,  1342. 

U.  S.  Senate  Document  No.  469,  60th  Congress,  1st  Session.  Plans 
for  International  Standards  for  Testing  Mineral  Oil  Products. 

1909.  DAY  and  GILPIN  :  Changes  in  Crude  Petroleum  effected  by  Diffusion 

through  Clay.     /.  Ind.  Eng.  Chem.,  1,  449. 
GILLETT  :  Analyses    and    Friction    Tests   of    Lubricating    Greases. 

J.  Ind.  Eng.  Chem.,  1,  351. 

HYDE  :  Definition  of  Gasoline.     J.  Ind.  Eng.  Chem.,  1,  377. 
LADD  :  Experiments  with  Burning  Oils.     N.  Dak.  Agl.  Expt.  Sta.r 

18th  Ann.  Report,  pp.  34-43 ;  Chem.  Abs.,  3,  243. 
MAGRUDER  :  Rapid  Method  for  the  Determination  of   Sulphur  in 

Crude  Petroleum.     Chem.  Abs.,  3,  115. 
SADTLER  :  (Methods  of  avoiding  Emulsions  in  Extraction  of  tTnsa- 

ponifiable  Oil).     J.  Ind.  Eng.  Chem.,  1,  479. 
SHERMAN,  GRAY,  and  HAMMERSCHLAG  :  Comparison  of  Calculated 

and  Determined  Viscosity  Numbers  and  Flashing  and  Burning 

Points  in  Oil  Mixtures.     /.  Ind.  Eng.  Chem.,  1,  13. 
STORMER  :  Viscosimeter.     /.  Ind.  Eng.  Chem.,  1,  317. 
UBBELOHDE  :  Viscosity  of  Illuminating  Oils  and  an  Apparatus  for  its 

Determination.     Petroleum,  1909,  4,  861 ;  Chem.  Abs.,  3,  2376. 


238  METHODS   OF   ORGANIC    ANALYSIS 

1910.  CONRADSON  :  Laboratory    Tests    of    Lubricants  —  Interpretation   of 

Analyses.     /.  Ind.  Eng.  Chem.,  2,  171. 
Goss :  Oils  and  Lubricants.     Modern  Power,  1,  No.  4;  Chem.  Abs., 

5,  379. 
KISSLING  :  Examination   of  Crude  Petroleum  and  of  its  Products. 

Petroleum,  1910,  5,  505;  Chem.  Abs.,  4,  1365. 
KISSLING  :  Determination  of  Asphalt  in  Cylinder  Oils.     Chem.  Rev. 

Fett-  Harz-Ind.,  17,  35;   Chem.  Abs.,  4,  1367. 

MABERY:  Lubrication  and  Lubricants.     J.  Ind.  Eng.  Chem.,  2,  115. 
MEISSNER:  Influence  of  Errors  in  the  Dimensions  of  Engler's  Yis- 

cosimeter.     Chem.  Rev.  Fett- Harz-Ind.,  17,  202;    Chem.  Abs.,  4, 

3148. 
ROBERTS  and  FRASER  :  Estimation  of  Water  in  Petroleum.     /.  Soc. 

Chem.  Ind.,  29,  197. 
WATERS  :  Action  of  Sunlight  and  Air  upon  some  Lubricating  Oils. 

J.  Ind.  Eng.  Chem.,  2,  451. 

1911.  DAY:  The   Production  of  Petroleum  in  1910.      Published  by  U.S. 

Geological  Survey. 

GROSCHUFF  :  Solubility  of  Water  in  Benzene,  Petroleum,  and  Par- 
affin Oils.  Z.  Electrochem.,  17,  348 ;  Chem.  Abs.,  5,  2550. 

KESSLER  and  MATHIASON:  On  the  Interpolation  Method  of  Oil 
Analysis.  J.  Ind.  Eng.  Chem.,  3,  66. 

LOEBELL:  Determination  of  Asphaltum  Insoluble  in  a  Mixture  of 
Alcohol  and  Ether  in  Mineral  Lubricating  Oils.  Petroleum,  6, 
774;  Chem.  Abs.,  5,  3149. 

WATERS  :  The  Effect  of  Added  Fatty  and  Other  Oils  upon  the  Car- 
bonization of  Mineral  Lubricating  Oils.  J.  Ind.  Eng.  Chem.,  3, 
812. 


CHAPTER   XII 
Fuels 

THE  purpose  of  this  chapter  is  to  outline  the  direct  determi- 
nation of  the  calorific  value  of  solid  and  liquid  fuels  and  then 
to  consider  the  analytical  determinations  of  most  importance 
for  the  judgment  of  each  of  the  chief  types  of  fuel  and  especi- 
ally the  relation  of  the  chemical  composition  to  the  calorific 
power. 

DETERMINATION  OF  CALORIFIC  POWER 

The  heat  of  combustion  or  calorific  power  of  a  solid  or 
liquid  fuel  is  best  determined  by  burning  in  oxygen  in  a  bomb 
calorimeter  according  to  the  general  method  of  Berthelot.  The 
chief  modifications  of  the  Berthelot  bomb  in  use  in  this  country 
are  those  of  At  water,  Emerson,  and  Mahler. 

The  Atwater  apparatus  has  been  fully  described  by  Atwater 
and  Snell  in  the  Journal  of  the  American  Chemical  Society  for 
July,  1903.  The  original  description  of  the  Emerson  calori- 
meter will  be  found  in  the  Journal  of  Industrial  and  Engineering 
Chemistry  for  January,  1909,  and  the  Mahler  bomb  is  well  de- 
scribed by  Gill  in  his  Gras  and  Fuel  Analysis  for  Engineers. 
Since  these  descriptions  as  well  as  others  referred  to  at  the  end 
of  this  chapter  are  readily  accessible  and  full  directions  are  usu- 
ally furnished  with  the  apparatus  by  the  manufacturer,  it  is  not 
so  important  here  to  discuss  the  exact  details  of  manipulation, 
which  depend  to  some  extent  upon  the  form  of  bomb  calorimeter 
used,  as  to  outline  certain  principles  and  precautions  of  general 
application. 

The  heat  of  combustion  is  determined  by  burning  in  the 
bomb  in  an  atmosphere  of  oxygen  a  small  amount  of  the  sub- 

239 


240  METHODS    OF   ORGANIC    ANALYSIS 

stance,  usually  enough  to  yield  about  5  or  6  large  calories,  and 
measuring  the  rise  of  temperature  of  the  known  amount  of 
water  in  which  the  bomb  is  immersed.  In  order  to  be  able  to 
determine  the  quantity  of  heat  from  the  rise  in  temperature  it 
is  necessary  to  know  not  only  the  amount  of  water  surround- 
ing the  bomb  but  also  the  heat  capacity  of  the  apparatus.  There 
are  four  general  methods  of  estimating  this  heat  capacity : 

(1)  By  calculation  from  the  weights  and  specific  heats  of  the 
materials  of  which  the  apparatus  is  composed. 

(2)  By  some  application  of  the  "  method  of  mixtures,"  such 
as  placing  the  bomb  in  a  known  amount  of  water  and  after  the 
temperature  has  become  uniform  adding  a  known  amount  of 
water  of  a  different  (known)  temperature  and  noting  the  change 
of  temperature  of  the  system. 

(3)  By  burning  in  the   bomb  under  the  conditions  of  an 
ordinary  determination  a  weighed  amount  of  substance  whose 
heat  of  combustion  is  accurately  known  and  noting  the  rise  of 
temperature  produced  by  this  known  amount  of  heat. 

(4)  By  generating  a  known  amount  of  heat  in  the  bomb 
electrically  and  measuring  the  resulting  rise  of  temperature. 

The  first  method  is  not  sufficiently  accurate  for  final  calibra- 
tion of  a  bomb  but  is  sometimes  useful  for  a  preliminary  calcu- 
lation of  approximate  heat  capacity. 

The  second  method  is  accurate  only  when  carried  out  under 
conditions  and  with  precautions  so  troublesome  as  to  be  prac- 
tically prohibitive. 

The  third  method  is  more  accurate  than  the  first  or  second, 
and  is  the  one  now  generally  used,  but  it  involves  the  assump- 
tion that  the  heat  of  combustion  of  the  "standard  substance" 
is  accurately  known. 

The  fourth  method l  is  the  most  accurate  but  is  hardly  neces- 
sary except  as  an  ultimate  basis  in  the  preparation  of  standard 
substances. 

The  Bureau  of  Standards  recommends  that  calorimeters 
generally  be  calibrated  by  the  third  method  using  standard 

1  Jaeger  and  Steinmehr:  Ann.  d.  Phys.,  21,  23-63  (1906)  and  Bulletin  of 
the  Bureau  of  Standards,  Reprint  No.  135. 


FUELS  241 

combustion  samples,  the  heats  of  combustion  of  which  have 
been  carefully  determined  in  calorimeters  calibrated  by  elec- 
trical means. 

The  heat  capacity  of  the  apparatus  is  usually  expressed  as 
its  "water  equivalent"  or  "  hydrothermal  equivalent."  Thus 
a  "  water  equivalent  "  or  "  hydrothermal  equivalent "  of  408 
would  mean  that  the  heat  capacity  of  the  apparatus  was  equal 
to  that  of  408  grams  of  water. 

The  specific  heat  of  water  changes  somewhat  with  the  tem- 
perature so  that  a  slight  error  is  introduced  if  a  calorie  be 
taken  in  one  case  as  the  heat  capacity  of  water  per  degree 
centigrade  at  zero  and  in  another  case  at  room  temperature. 
The  Bureau  of  Standards  adopts  the  heat  capacity  of  water  at 
15°  C.  as  unity  so  that  the  calorie  is  defined  as  the  heat  capacity 
of  one  gram  of  water  per  degree  centigrade  at  a  temperature 
of  15°  C.  and  the  B.  T.  U.  as  the  heat  capacity  of  one  pound  of 
water  per  degree  Fahrenheit  at  a  temperature  of  60°  F.  For- 
tunately it  is  not  necessary  in  ordinary  determinations  of  heat 
of  combustion  to  change  the  basis  of  calculation  with  the  tem- 
perature of  working  between  15°  and  25°  C.,  because  the 
Bureau  of  Standards  has  found  that  the  total  heat  capacity  of 
an  ordinary  combustion  calorimeter  does  not  change  apprecia- 
bly with  temperature  between  15°  and  25°  C.  This  is  due  to 
the  fact  that  the  metal  of  the  bomb  and  accessories  has  a  posi- 
tive temperature  coefficient,  while  the  water  has  a  negative 
temperature  coefficient  throughout  this  range  of  temperature 
and  the  two  nearly  neutralize  each  other. 

Standard  Materials.  —  Among  the  substances  which  have 
been  used  in  the  standardization  of  combustion  calorimeters 
are  cane  sugar,  benzoic  acid,  naphthalene,  glycocoll,  hippuric 
acid,  and  camphor.  The  Bureau  of  Standards  recommends  the 
first  three  and  supplies  standard  samples  of  these  for  this  pur- 
pose. 

Sucrose  is  not  volatile  nor  strongly  hygroscopic,  but  is 
rather  difficult  to  ignite,  sometimes  fails  to  burn  completely, 
and  has  a  heat  of  combustion  only  about  half  as  high  as  that  of 
good  coal. 


242  METHODS  OF  ORGANIC  ANALYSIS 

Benzole  acid  is  only  slightly  volatile,  not  very  hygroscopic, 
burns  readily,  and  has  a  heat  of  combustion  about  four-fifths 
that  of  ordinary  coal. 

Naphthalene  is  more  volatile  than  benzoic  acid,  but  is  not 
hygroscopic,  burns  very  readily,  and  has  a  heat  of  combustion 
a  little  higher  than  that  of  coal.  The  Bureau  of  Standards 
reports  that  the  loss  by  sublimation  from  naphthalene  pressed 
into  pellets  for  combustion  will  hardly  exceed  0.1  to  0.2  per 
cent  in  an  hour,  so  that  any  error  in  standardization  due  to 
volatility  of  the  naphthalene  should  be  less  than  0.1  per  cent. 

Although  sugar  has  been  most  used  in  the  past,  the  Bureau 
of  Standards  finds  benzoic  acid  the  most  satisfactory  substance 
for  accurate  standardization  of  bomb  calorimeters. 

Preparation  of  sample  or  charge.  —  Since  the  amount  of  mate- 
terial  used  for  a  determination  is  usually  not  over  a  gram,  the 
sample  must  be  finely  ground  and  thoroughly  mixed.  A  por- 
tion of  the  mixed  powdered  sample  is  then  pressed  into  a  pellet 
or  small  briquet,  which -is  weighed  in  the  combustion  crucible 
and  then  placed  in  the  bomb.  Pressing  the  charge  into  a  pel- 
let avoids  the  danger  of  portions  being  blown  out  of  the  cruci- 
ble either  when  the  oxygen  is  admitted  to  the  bomb  or  during 
the  rapid  combustion  which  takes  place  when  the  substance  is 
fired.  In  standardizing  with  napthalene  the  loss  by  volatiliza- 
tion is  also  much  smaller  from  a  pellet  than  from  the  same 
weight  of  loose  material.  Liquids,  and  anthracite  coals  which 
cannot  be  pressed  into  pellets,  are  conveniently  burned  in 
weighed  hard  "  gelatin "  capsules  of  determined  calorific 
power.  The  objections  sometimes  offered  to  this  method 
are  probably  due  to  the  use  of  capsules  of  unsuitable  char- 
acter. The  writer  uses  Parke-Davis  capsules  of  "five-grain" 
size  which  weigh  about  one  tenth  gram  each,  have  a  heat  of 
combustion  of  about  4480  calories  per  gram,  are  neither  sticky 
nor  hygroscopic,  and  burn  readily,  thus  aiding  the  complete 
combustion  of  high-ash  anthracite  coals.  Some  prefer  to  burn 
anthracites  in  a  loose  condition  and,  if  difficulty  is  experienced 
in  obtaining  complete  combustion,  to  place  beneath  the  anthra- 
cite a  weighed  amount  of  bituminous  coal  of  known  calorific 


FUELS  243 

power.  Blakeley  and  Chance  hold  that  the  failure  of  high-ash 
coals  to  burn  completely  when  placed  directly  in  the  combus- 
tion crucible  is  largely  due  to  the  cooling  of  that  portion  of  the 
coal  which  is  in  contact  with  the  metal  floor  of  the  crucible  and 
may  be  avoided  by  placing  a  disk  of  asbestos  beneath  the  coal. 

Pasty  solids  and  non-volatile  liquids  may  be  weighed  and 
burned  directly  in  the  combustion  crucibles.  In  the  case  of 
fatty  oils  it  was  found  advantageous  to  add  a  little  loose  as- 
bestos (previously  ignited)  to  regulate  the  burning  and  pre- 
vent possible  loss  by  spattering. 

For  a  volatile  liquid  select  a  capsule  having  a  body  with  a 
smooth  edge  and  a  snug-fitting  cap,  transfer  the  liquid  from 
the  sample  bottle  to  the  weighed  capsule  by  means  of  a  pipette 
or  medicine  dropper,  close  the  capsule,  stand  it  in  the  combus- 
tion crucible  and  weigh,  then  allow  to  stand  about  5  minutes 
and  weigh  again  to  make  sure  that  none  of  the  sample  is  being 
lost  by  evaporation.  Should  it  be  found  that  the  capsule  per- 
mits loss  by  evaporation,  it  must  be  rejected  and  another  pre- 
pared and  tested  in  the  same  way. 

Both  in  standardizing  the  bomb  and  in  the  testing  of  fuels 
the  amount  of  the  charge  should  depend  upon  its  calorific 
power  so  that  there  may  not  be  great  differences  in  the  amount 
of  heat  to  be  measured  in  the  different  determinations.  When 
the  charge  has  been  prepared  in  the  combustion  crucible,  it  is 
placed  in  position,  the  fuse  wire  adjusted,  the  bomb  closed  and 
charged  with  oxygen. 

The  oxygen  used  for  combustion  must  be  the  purest  obtainable 
commercially,  especially  as  regards  freedom  from  combustible 
gases  such  as  hydrogen,  carbon  monoxide,  and  hydrocarbons. 
Each  cylinder  of  oxygen  should  be  tested  by  determinations 
upon  standard  combustion  samples  such  as  are  used  in  deter- 
mining the  heat  capacity  of  the  apparatus. 

The  bomb  is  charged  with  oxygen  to  a  pressure  of  about  25 
atmospheres,  and,  after  being  properly  closed  to  prevent  any  es- 
cape of  gas,  is  immersed  in  a  weighed  quantity  of  water,  the 
terminals  of  the  firing  circuit  adjusted,  the  covers  placed  in 
position,  and  the  stirring  and  thermometric  observations  begun. 


244          METHODS  OF  ORGANIC  ANALYSIS 

The  details  of  these  operations  vary  with  the  different  forms  of 
apparatus  and  are  explained  for  each  in  the  descriptions  already 
cited. 

At  the  end  of  the  "  fore-period  "  in  which  the  rate  of  change 
due  to  the  temperature  of  the  surroundings  has  been  determined, 
the  charge  is  ignited  by  closing  the  switch  of  the  firing  circuit 
so  as  to  pass  through  the  fuse  wire  a  current  sufficient  to  heat 
it  to  redness  if  platinum,  or  cause  it  to  burn  if  iron,  but  of  low 
potential  so  as  to  avoid  danger  of  arcing  with  evolution  of  heat 
within  the  bomb. 

Temperature  measurements  should  be  made  with  delicate  dif- 
ferential thermometers  of  the  Beckmann  type  which  should  be 
carefully  calibrated,  preferably  by  the  Bureau  of  Standards  or 
the  Reichsanstalt.  Accuracy  requires  that  the  proper  correc- 
tions be  determined  and  applied  for  errors  of  the  scale,  for  the 
emergent  stem,  and  for  the  amount  of  mercury  removed  from 
the  bulb.  The  first,  called  "  caliber  correction,"  should  be  ap- 
plied to  each  reading  ;  the  second  and  third  together  are  often 
called  "  thermometer  correction,"  and  this  correction,  which 
will  depend  upon  the  room  temperature  and  the  setting  of  the 
thermometer,  is  applied  to  the  apparent  rise  of  temperature  ob- 
served during  the  combustion,  to  obtain  the  rise  in  true  degrees. 

The  radiation  correction  is  calculated  from  the  readings  taken 
before,  during,  and  after  the  combustion,  preferably  by  means 
of  the  Regnault-Pfaundler  formula. 

At  the  end  of  the  after-period  following  a  combustion  the 
bomb  is  opened  and  a  careful  examination  made  for  any  evi- 
dences of  incomplete  combustion.  Any  portions  of  the  fuse- 
wire  which  may  remain  unburned  are  weighed  and  deducted 
from  the  original  weight.  The  bomb  is  rinsed  out  and  the 
nitric  acid  which  has  been  formed  during  the  combustion  is 
determined  by  titration.  The  heat  of  formation  and  solution 
of  the  nitric  acid  formed  (230  calories  per  gram),  as  well  as  the 
heat  of  combustion  of  the  iron  fuse  wire  burned  (1600  calories 
per  gram),  and  of  the  gelatin  capsule  if  used,  must  be  allowed 
for  in  the  calculation  of  the  results.  The  full  explanation  of  the 
calculation  of  results  given  by  Atwater  and  Snell  should  be 


FUELS  245 

consulted.  Fuels  containing  sulphur  yield  sulphuric  acid  on 
combustion  in  the  bomb,  so  that  a  slight  error  is  introduced  if 
the  total  acidity  of  the  bomb  rinsings  be  calculated  as  nitric 
acid.  To  correct  for  this  the  usual  subtraction  for  total  acidity 
calculated  as  nitric  acid  may  be  made,  then  the  sulphuric  acid 
in  the  rinsings  may  be  found  by  precipitation  and  the  correction 
increased  by  13  calories  per  gram  for  each  per  cent  of  sulphur 
in  the  fuel.1  This  is  based  on  the  assumption  that  as  fuel  is 
ordinarily  used  tjie  sulphur  burns  to  sulphur  dioxide. 

The  calorific  powers  as  found  in  the  bomb  calorimeter  repre- 
sent somewhat  more  heat  than  is  actually  obtained  from  the  fuel 
in  ordinary  use,  not  only  because  the  combustion  is  complete, 
but  especially  because  the  water  vapor  produced  in  the  combus- 
tion condenses  in  the  bomb  and  the  latent  heat  of  this  vapor  is 
not  lost  as  is  usually  the  case  when  the  fuel  is  burned  in  use. 
Occasionally  an  attempt  is  made  to  distinguish  between  "  heat 
of  combustion  "  and  "  calorific  power,"  confining  the  latter  term 
to  the  values  obtained  on  subtracting  from  the  heat  of  com- 
bustion the  estimated  heat  of  vaporization  of  the  water.  More 
often  the  heat  of  combustion  is  called  the  "  calorific  power  (high 
value)  "  or  simply  "  calorific  power,"  and  the  term  "  calorific 
power  (low  value)"  is  used  to  designate  the  value  obtained 
on  deducting  the  latent  heat  of  water  vapor.  In  this  chapter 
the  "high  values"  are  always  given  without  the  use  of  that 
term,  or  in  other  words  the  term  calorific  power  is  used  as 
synonymous  with  heat  of  combustion. 

CHEMICAL  COMPOSITION  AND  CALORIFIC  POWER  OF 
ORGANIC  COMPOUNDS 

According  to  the  usually  accepted  determinations,  the  heat  of 
combustion  of  carbon  is  8080,  and  of  hydrogen  34,500,  calories 
per  gram.  The  heat  of  combustion  of  a  compound  consisting 
of  carbon  and  hydrogen  only  is  the  sum  of  the  heats  of  combus- 
tion of  the  carbon  and  hydrogen  it  contains  minus  the  heat  of 
formation  of  the  compound,  which  is  for  hydrocarbons  a  rela- 

1  Lord  :  U.  S.  Geological  Survey,  Professional  Paper  No.  48. 


246  METHODS    OF   ORGANIC    ANALYSIS 

lively  small  factor.  In  compounds  containing  oxygen  the  heat 
of  formation  is  larger  and  the  heat  of  combvistion  is  proportion- 
ately less  than  the  heat  which  would  be  obtained  by  burning  the 
quantities  of  carbon  and  hydrogen  present,  for  the  obvious  rea- 
son that  these  elements  are  already  partially  "  oxidized  "  by  the 
oxygen  present  in  the  molecule.  From  the  heats  of  combustion 
of  carbon  and  hydrogen  one  may  readily  calculate  that  a  given 
weight  of  oxygen  will  cause  a  greater  evolution  of  heat  in  burn- 
ing hydrogen  to  water  than  in  burning  carbon  to  carbon  di- 
oxide. Hence  in  the  case  of  a  compound  of  carbon,  hydrogen, 
and  oxygen,  the  estimated  calorific  power  will  be  lower  if  we 
assume  that  the  oxygen  in  the  molecule  is  to  be  considered  as 
combined  with  hydrogen  than  if  we  assume  that  it  is  to  be  con- 
sidered as  combined  with  carbon. 

Usually  estimates  of  calorific  power  from  ultimate  analysis 
have  been  based  on  "  Welter's  rule,"  which  assumes  that  the 
oxygen  present  is  combined  with  hydrogen,  or,  that  we  shall 
offset  the  heat  of  formation  if  we  deduct  the  quantity  of  heat 
which  would  be  produced  if  the  oxygen  present  combined  with 
the  hydrogen.  On  this  assumption  the  heat  of  combustion  of 
such  a  compound  would  be  represented  by  the  formula: 

X  =  8080  C  +  34,500  (H  -  -J  O). 

Walker,  in  his  Introduction  to  Physical  Chemistry,  pointed 
out  that  the  values  thus  obtained  are  considerably  too  low  in 
the  cases  of  sugar  and  of  butyric  acid,  and  stated  that  "  a  better 
result  may  usually  be  obtained  by  subtracting  the  oxygen,  not 
with  the  corresponding  quantity  of  hydrogen,  but  with  the 
corresponding  quantity  of  carbon,  and  then  estimating  the  heat 
of  combustion  of  the  elements  in  the  residue."  Putting  this 
suggestion  (which  for  convenience  we  may  call  "  Walker's  rule  " 
as  contrasted  with  "  Welter's  rule  ")  in  the  form  of  an  equation, 
we  have, 

X  =  8080  (C  -  §  O)  +  34,500  H. 

If,  now,  these  two  formulae  be  applied  to  the  various  classes 
of  pure  organic  compounds,  and  the  results  compared  with  the 
experimentally  determined  heats  of  combustion  as  compiled  by 


FUELS  247 

Berthelot,  or  Vaubel,  it  will  be  found  that  in  some  cases  the 
higher,  in  other  cases  the  lower,  of  the  calculated  results  ap- 
proximates the  true  value,  while  for  still  other  compounds  the 
true  value  lies  between  them  and  at  a  distance  from  either. 
The  relative  values  of  these  formulae  as  applied  to  different 
classes  of  fuels  is  considered  below. 

In  some  cases  fuel  values  are  estimated  from  proximate, 
rather  than  ultimate,  chemical  composition.  Thus,  since  the 
calorific  power  of  pure  alcohol  and  the  relation  of  specific  gravity 
to  percentage  strength  of  alcohol-water  mixtures  are  both 
known,  the  specific  gravity  of  any  commercial  alcohol  which  is 
essentially  a  mixture  of  pure  alcohol  and  water  will  show  the 
amount  of  alcohol  present,  and  hence  the  calorific  power  of  the 
liquid.  In  food  analysis  the  fuel  value  is  commonly  estimated 
from  the  percentages  of  proteins,  fats,  and  carbohydrates  as  has 
been  fully  explained  in  another  volume.1 

FUEL  OILS  AND  GASOLINE 

With  appliances  sufficiently  well  adapted  to  its  properties, 
any  petroleum  oil  can  be  burned  with  an  evolution  of  heat  much 
greater  than  that  of  any  coal. 

Since  American  petroleums  are  essentially  mixtures  of  hydro- 
carbons and  chiefly  of  the  methane  series,  it  was  to  be  expected 
that  the  greater  the  specific  gravity  of  the  sample  the  greater 
would  be  the  mean  molecular  weight  and  percentage  of  carbon 
and  the  less  would  be  the  percentage  of  hydrogen  and  the  heat 
of  combustion  per  gram.  In  general  a  specific  gravity  of 

0.7  -0.75  indicates  about  11,500-11,300  calories  per  gram  ; 
0.75-0.8  indicates  about  11,300-11,100  calories  per  gram  ; 
0.8  -0.85  indicates  about  11,100-10,900  calories  per  gram  ; 
0.85-0.9  indicates  about  10,900-10,700  calories  per  gram  ; 
0.9  -0.933  indicates  about  10,700-10,500  calories  per  gram. 

More  commonly  in  English-speaking  countries  the  density  of 

petroleum  oils  is  stated  in  terms  of  the  Baume  scale  and  the 

1  Sherman  :  Chemistry  of  Food  and  Nutrition,  Chapter  IV. 


248 


METHODS   OF   ORGANIC   ANALYSIS 


calorific  power  in  British  thermal  units.     The  relations  of  the 
values  are  as  follows  : 

calories  per  gram  x  1.8  =  B.  T.  U.  per  pound  ; 

•«  •* 

specific  gravity 


From  the  densities  and  calorific  powers  of  86  samples  of 
American  petroleum  oils,  70  of  which  were  examined  by  the 
writer  and  his  associates  at  Columbia  University,  and  16  by 
Allen  and  Strong  of  the  U.  S.  Geological  Survey,  is  constructed 
the  following  table  1  for  estimating  the  calorific  power  from  the 
Baume  density  in  commercially  pure  petroleum  oils. 

TABLE  20.  —  APPROXIMATE  CALORIFIC  POWERS,  IN  BRITISH   THERMAL 
UNITS  PER  POUND,  OF  PETROLEUM  OILS  OF  20°  to  67°  BAUME 


Density 
degrees 
Baume 

Calorific  power 
B.  T.  U. 
per  pound 

Density 
degrees 
Baume 

Calorific  power 
B.  T.  U. 
per  pound 

Density 
degrees 
Baum6 

Calorific  power 
B.  T.  U. 
per  pound 

20 

18930 

36 

19735 

52 

20220 

21 

18990 

37 

19770 

53 

20245 

22 

19050 

38 

19805 

54 

20270 

23 

19110 

39 

19840 

55 

20290 

24 

19170 

40 

19875 

56 

20310 

.      25 

19225 

41 

19910 

57 

20330 

26 

19280 

42 

19940 

58 

20350 

27 

19335 

43 

19970 

59 

20370 

28 

19390 

44 

20000 

60 

20390 

29 

19445 

45 

20030 

61 

20410 

30 

19495 

46 

20060 

62 

20430 

31 

19545 

47 

20090 

63 

20450 

32 

19590 

•  48 

20120 

64 

20470 

33 
34 

19630 
19665 

49    , 
50 

20145 
20170 

65 
66 

20490 
20510 

35 

19700 

51 

20195 

67 

20530 

The  values  given  in  the  table  were  found  by  plotting  the  data 
of  all  the  samples,  drawing  a  smooth  curve  through  the  approxi- 
mate mean  results,  and  taking  from  this  curve  the  calorific 
power  (in  the  nearest  multiple  of  5  units)  corresponding  to  each 
degree  of  density  on  the  Baume*  scale.  On  taking  the  calorific 

1  Revision  of  the  work  of  Sherman  and  Kropff  (J.  Am.  Chem.  Soc.,  30, 1C26). 


FUELS  249 

powers  from  the  table  for  each  individual  sample  and  compar- 
ing with  the  value  determined  in  the  bomb  calorimeter,  it  was 
found  that  of  86  samples,  ranging  from  gasoline  to  the  heaviest 
crude  oils,  in  only  1  case  in  15  did  the  estimated  value  differ 
from  that  experimentally  determined  by  as  much  as  1  per  cent; 
in  only  1  in  43  was  the  difference  as  much  as  2  per  cent ;  in  no 
case  was  the  difference  as  much  as  3  per  cent. 

It  is  evident,  therefore,  that  in  commercially  pure  American 
petroleum  oils  the  calorific  power  may  be  estimated  from  the 
density  by  means  of  the  above  table  with  a  sufficient  degree  of 
accuracy  for  many  practical  purposes. 

Since  in  gasoline  engines,  and  in  some  of  the  machines  using 
other  fuel  oils,  the  combustion  is  preceded  by  vaporization  of 
the  fuel,  it  is  evident  that  volatility  is  an  important  factor  in 
determining  the  adaptability  of  the  gasoline  or  fuel  oil  to  the 
engine  in  which  it  is  to  be  used. 

In  general,  the  mechanical  engineer  judges  the  volatility 
from  the  density,  but  since  commercial  gasoline  or  other  fuel 
oil  is  a  mixture  of  numerous  lighter  and  heavier  hydrocarbons, 
"a  definite  constant  density  is  not  a  guarantee  that  the  compo- 
sition may  not  change  sufficiently  to  affect  the  action  of  the  fuel 
in  an  engine"  (Lucke  and  Woodward). 

In  order  to  determine  the  character  of  a  sample  in  this  re- 
spect, it  may  be  submitted  to  distillation  in  an  ordinary  distill- 
ing flask,  collecting  the  distillate  in  convenient  fractions  of  the 
volume  of  the  sample  taken,  and  noting  the  temperature  of  dis- 
tillation of  each  fraction  by  means  of  a  thermometer  so  placed 
that  the  top  of  the  mercury  bulb  is  on  a  level  with  the  bottom 
of  the  outlet  in  the  neck  of  the  distilling  flask,  so  as  to  show 
the  temperature  of  the  vapors  as  they  pass  from  the  flask  into, 
the  condenser. 

The  "  motor  gasoline  "  used  by  Lucke  and  Woodward  in  their 
comparison  of  alcohol  and  gasoline  as  fuel  for  internal  combus- 
tion engines  in  1906—07  was  examined  in  this  manner,  150  cc. 
being  distilled,  and  the  distillate  collected  in  fractions  of  10  cc. 
each,  with  the  following  results:  l 

1  U.  S.  Dept.  Agriculture,  Office  of  Experiment  Stations,  Bui.  191,  p.  23. 


250 


METHODS   OF   ORGANIC   ANALYSIS 


Number  of  fraction 

Temperature  of 
distillation  °  C. 

Number  of  fraction 

Temperature  of 
distillation  °  C. 

1 

46-60 

9 

100-104 

2 

64-75 

10 

104-108 

3 

75-80 

11 

108-112 

4 

80 

12 

112-120 

5 

80-86 

13 

120-126 

6 

86-92 

14 

126-140 

7 
8 

92-97 
97-100 

15  (5  cc.) 

140-155 

Residue  at  155°  C.  =  5  cc.  or  3.3  pet. 

A  sample  purchased  in  New  York  City  in  1908,  and  believed 
to  be  a  representative  specimen  of  satisfactory  automobile  gaso- 
line, was  examined  by  the  writer  with  the  following  results: 

(1)     DISTILLATION  OF  300  cc.  IN  FRACTIONS  ACCORDING  TO  VOLUME 


Number  of  fraction 

Temperature  of 
distillation  °  C. 

Volume  cc. 

Specific  Gravity  at 
15°  C. 

1 

40-68 

30 

0.669 

2 

68-74 

30 

0.678 

3 

74-81 

30 

0.692 

4 

81-86 

30 

0.704 

5 

86-91 

30 

0.712 

6 

91-96 

30 

0.720 

7 

96-102 

30 

0.727 

8 

102-110 

30 

0.734 

9 

110-121 

30 

0.741 

Residue  at  121°  C. 

30 

0.756 

(2)     DISTILLATION  OF  300  cc.  IN  FRACTIONS  ACCORDING  TO 
TEMPERATURE 


Number  of  fraction 

Temperature  of 
distillation  °  C. 

Volume 

Specific  gravity 
at  15°  C. 

cc. 

% 

1 

40-70 

40.5 

13.5 

0.670 

2 

70-80 

47.5 

15.8 

0.690 

3 

80-90 

60. 

20. 

0.706 

4 

90-100 

47.5 

15.8 

0.722 

5 

100-110 

42.5 

14.2 

0.733 

6 

110-120 

29.5 

9.9 

0.741 

7 

Residue  at  120°  C. 

0.755 

FUELS 


251 


These  data  may  be  useful  for  purposes  of  comparison  when 
examining  commercial  gasolines  with  reference  to  their  utility 
as  fuel  for  internal  combustion  engines. 

Three  samples  of  "fuel  oil"  purchased  by  gas  manufacturers 
in  New  York  City  in  1905  and  1906,  showed,  when  divided  into 
fifths  by  fractional  distillation,  the  following  results : 

TABLE  21.  —  COMPARISON  OF  FRACTIONS  OF  COMMERCIAL  "FUEL  OILS' 


Temperature  of 
Distillation 

°C. 

Density  at  15.5°  C.  (60°  F.) 

Specific 
Gravity 

Degrees 
Baume 

Sample  A. 
1st  fraction 

165-257 
257-290 
290-318 
318-340 

138-243 
243-288 
288-327 
327-353 

160-260 
260-286 
286-305 
305-330 

0.832 
0.870 
0.880 
0.886 
0.900 

0.770 
0.851 

0.871 
0.883 
0.906 

0.795 
0.843 
0.853 

0.861 
0.881 

38.3 
30.9 
29.1 
28.0 
25.5 

51.8 
34.5 
30.7 
28.5 
24.5 

46.1 
36.1 
34.1 
32.6 
28.9 

2d  fraction      

3d  fraction      

4th  fraction     

Residue  

Sample  B. 
1st  fraction      

2d  fraction 

3d  fraction 

4th  fraction 

Sample  C. 

3d  fraction       

4th  fraction     .... 

Residue  

WOODS  AND  SIMILAR  FUELS 

Wood,  peat,  spent  tan,  bagasse,  and  other  similar  materials  used 
as  fuel  differ  greatly  in  calorific  power  because  of  wide  fluctua- 
tions in  moisture  and  ash  content  and  considerable  differences 
in  the  nature  of  their  organic  constituents;  for  instance,  some 
woods  approximate  cellulose,  while  others  contain  large  amounts 


252 


METHODS  OF  ORGANIC  ANALYSIS 


of  resinous  material  of  much  higher  calorific  power.  All  the 
fuels  of  this  general  group  are,  however,  characterized  by  high 
oxygen  content  and  low  heat  of  combustion  as  compared  with 
good  coal,  and  so  in  attempts  to  estimate  the  calorific  power  from 
the  ultimate  analysis  the  differences  between  the  results  obtained 
by  the  use  of  Welter's  rule  or  Walker's  suggestion  as  explained 
above  are  relatively  larger  for  this  group  of  fuels  than  for  coals. 

In  order  to  ascertain  which  method  of  calculation  is  preferable 
and  whether  either  yields  accurate  results,  Sherman  and  Amend 
analyzed  eight  fuels  of  this  type  and  compared  the  results  obtained 
by  each  method  of  calculation  with  those  actually  determined 
by  combustion  in  oxj^gen. 

The  results  of  ultimate  chemical  analysis,  reduced  to  the  basis 
of  dry  matter  were  as  follows: 

TABLE  22.  —  ULTIMATE  COMPOSITION  OF  DRY  MATTER  OF  WOOD,  ETC. 


Sample 

Carbon 

Hydrogen 
% 

Oxygen 

Nitrogen 

Sulphur 

Ash 

Chestnut  wood  chips     .     .     . 

50.28 

5.58 

43.21 

0.10 

0.03 

0.80 

Chestnut  wood  chips,  leached 

50.09 

5.65 

43.33 

0.10 

0.02 

0.81 

Hemlock  tan    ...... 

53.74 

5.66 

39.05 

0.24 

0.04 

1.27 

Hemlock  tan,  leached    .     .     . 

54.97 

5.73 

37.69 

0.26 

0.02 

1.33 

Oak  tan,  leached  

49.51 

5.53 

39.24 

0.40 

0.05 

5.27 

Bagasse   

49.04 

5.96 

42.58 

.  0.31 

0.07 

2.04 

"  Oil  cake  "  .                   ... 

48.90 

6.47 

38.02 

4.24 

0.25 

2.82 

Peat     ...                   .     . 

57.30 

4.66 

19.26 

1.13 

0.77 

16.88 

From  these  analyses  the  calorific  power  or  heat  of  combustion 
was  estimated  (1)  according  to  Dulong's  formula 

X=  8080 C  +  34,500 (H  -  JO)  +  2250 S, 

based  on  Welter's  rule  of  calculating  the  oxygen  with  the  hydro- 
gen, (2)  according  to  Walker's  suggestion  of  calculating  the 
oxygen  with  the  carbon.  Using  here  the  same  calorific  values 
for  the  elements  as  in  Dulong's  formula,  we  have  the  formula: 

X  =  8080  (C  -  |  O)  +  34,500  H  +  2250 S. 


FUELS 


253 


which  must  of  course  be  applied  to  the  percentages  in  the  water- 
free  substance. 

The  results  (in  calories)  obtained  by  these  two  formulae 
along  with  those  determined  directly  by  means  of  the  Atwater- 
Mahler  bomb  calorimeter  were  as  follows  : 


TABLE  23.  —  ESTIMATED  AND  DETERMINED  CALORIFIC  POWERS 


Welter's 
rule 

Walker's 
rule 

Direct 
determination 

Chestnut  wood  chips 

4125 

4659 

4632 

Chestnut  wood  chips,  leached     .     . 

4129 
4612 

4684 
5113 

4661 
5106 

4794 

5277 

5217 

Oak  tan  leached  .               .... 

4217 

4720 

4798 

Basrasse                                      .     .     . 

4185 

4731 

4625 

4594 

5078 

4953 

Peat     

5422 

5671 

5606 

Averagre          

4510 

4992 

4950 

It  will  be  seen  that  in  the  case  of  these  woody  fuels  high,  in 
oxygen  the  calorific  powers  estimated  by  means  of  Dulong's 
formula  based  on  Welter's  rule  are  all  much  too  low,  the  esti- 
mated values  in  terms  of  the  actual  ranging  from  88.4  to  96.7 
per  cent  and  averaging  90.4  per  cent,  while  the  values  estimated 
by  a  corresponding  formula  based  on  Walker's  rule  show  a  fair 
approximation  to  the  values  actually  determined  by  the  bomb 
calorimeter,  the  estimated  values  in  terms  of  the  determined 
ranging  from  100.1  to  102.5  per  cent  and  averaging  100.8  per 
cent. 

The  two  methods  of  calculation  were  also  applied  to  seven 
analyses  of  lignites  taken  from  Bulletin  290  of  the  U.  S.  Geo- 
logical Survey.  In  one  of  the  seven  cases  the  calorific  power 
actually  determined  was  lower  than  that  found  by  Welter's 
rule ;  in  one  it  was  materially  higher  than  that  found  by 
Walker's  rule ;  in  each  of  the  other  five  cases  and  also  in  the 
average  of  all  seven  the  results  by  Welter's  rule  were  much  too 


254  METHODS   OF   ORGANIC   ANALYSIS 

low  and  those  calculated  according  to  the  suggestion  of  Walker 
were  approximately  correct. 

The  results  indicate :  (1)  that  too  much  reliance  should 
not  be  placed  upon  estimates  of  calorific  power  from  ultimate 
chemical  composition,  especially  in  fuels  high  in  oxygen ; 
(2)  that  Dulong's  formula  or  any  simular  formula  based  on 
"  Welter's  rule  "  of  calculating  the  oxygen  with  the  hydrogen 
is  likely  to  give  results  much  below  the  truth;  (3)  that  the 
higher  results  obtained  by  calculating  the  oxygen  of  the  water- 
free  sample  as  combining  with  the  carbon  according  to  the 
suggestion  of  Walker  are  much  more  nearly  correct,  and  in 
most  cases  show  a  fair  approximation  to  the  values  determined 
directly. 

COAL 

Ultimate   Composition  and  Calorific  Power 

The  data  above  given  having  shown  that  the  calorific  power 
of  wood  and  similar  fuels  is  related  to  the  ultimate  composition 
much  more  nearly  according  to  Walker's  than  according  to 
Welter's  rule,  a  similar  comparison  of  these  two  methods  of 
calculating  calorific  power  was  made  for  coal.1  For  this  pur- 
pose the  analyses  and  calorific  powers  of  67  coals  examined 
by  the  U.  S.  Geological  Survey  and  described  in  Professional 
Paper  No.  48  were  used.  These  data  were  determined  in 
connection  with  the  studies  made  by  the  Survey  in  its  coal- 
testing  plant  at  the  St.  Louis  Exposition  in  1904  and  cover 
coals  mined  in  17  states  and  differing  widely  in  composition 
and  character. 

A  comparison  of  the  calorific  powers  as  calculated  by 
Dulong's  formula  based  on  Welter's  rule  with  those  found  by 
actual  determination  in  a  bomb  calorimeter  showed  that  the 
values  calculated  by  this  formula  were  below  those  found  by 
the  calorimeter  in  seven  eighths  of  the  cases  and  averaged  98.9 
per  cent  of  the  determined  values.  In  more  than  half  of  the 
cases  the  calculated  differed  from  the  determined  values  by 

1  Sherman,  Bartlett,  and  Weatherless ;  results  not  yet  published. 


FUELS  255 

over  1  per  cent ;  in  12  of  the  67  cases  or  about  1  in  6,  by  more 
than  2  per  cent ;  in  4  cases  or  about  1  in  17  by  more  than  3  per 
cent,  the  greatest  difference  being  3.8  per  cent. 

Thus  the  error  involved  in  the  use  of  the  Dulong  formula 
based  on  Welter's  rule  was  quite  variable.  The  values  thus 
calculated  averaged  1.1  per  cent  too  low,  but  it  is  evident  that 
the  calculated  results  of  individual  samples  cannot  be  made 
accurate  by  raising  them  all  by  a  corresponding  percentage ; 
for  the  extent  of  the  discrepancy  in  each  calculated  value  de- 
pends primarily  upon  the  oxygen  present  in  the  sample,  so  that 
to  increase  all  the  calculated  values  by  a  fixed  percentage 
would  give  calorific  powers  too  high  where  the  oxygen  content 
was  low  and  too  low  where  the  oxygen  content  was  high. 
Better  results  were  obtained  by  calculating  the  calorific 
powers  in  accordance  with  Walker's  suggestion  that  the 
oxygen  be  figured  as  combining  with  the  carbon  rather  than 
the  hydrogen. 

Calculating  the  calorific  powers  for  the  same  67  coals  accord- 
ing to  the  formula, 

X  =  8080  (C  -  f  O)  +  34,500  H  +  2250  S 

which  must  of  course  be  applied  to  the  percentages  in  the 
water-free  coal,  it  was  found  that  the  calculated  averaged  100.37 
per  cent  of  the  determined  values,  and  were  about  twice  as  often 
above  the  latter  as  below.  In  about  two  thirds  of  the  cases  the 
calculated  and  determined  values  agreed  within  1  per  cent,  and 
in  about  one  third  of  the  cases  differed  by  1  per  cent  or  more; 
6  of  the  67  cases,  or  about  1  in  11,  differed  by  more  than  2  per 
cent;  none  differed  by  as  much  as  3  per  cent.  Comparing  the 
results  obtained  by  the  two  methods  of  calculation  it  will  be 
seen  that  the  method  of  calculating  the  combined  oxygen  with 
the  carbon  instead  of  with  the  hydrogen  gives  a  much  better 
average  result,  a  smaller  proportion  of  cases  in  which  the  error 
exceeds  a  given  margin  (whether  this  be  one  or  two  per  cent) 
and  a  smaller  maximum  error. 

Of  the  formulae  at  present  available  for  calculating  the  calo- 
rific power  of  coal  from  its  ultimate  analysis  the  one  last  given 


256  METHODS   OF   ORGANIC    ANALYSIS 

should  therefore  be  used,  but  data  thus  calculated  are  much  less 
reliable  than  those  obtained  by  properly  conducted  determina- 
tions with  an  accurately  standarized  bomb  calorimeter. 

Proximate  Analysis  of  Coal 

The  proximate  analysis  of  coal  consists  in  determining  the 
moisture  and  ash,  and  separating  the  organic  matter  by  an 
arbitrary  heat  treatment  into  volatile  matter  and  fixed  carbon. 
In  order  that  results  of  different  analysts  may  be  comparable, 
a  uniform  method  has  been  agreed  upon  as  follows :  *  The 
sample  should  be  air-dried  and  ground  to  pass  a  100-mesh 
sieve.  • 

Moisture.  —  Dry  1  gram  of  the  fine-ground,  air-dry  sample  in 
an  open  platinum  or  porcelain  crucible  in  an  oven  at  104°  to 
107°  C.  for  1  hour ;  cool  in  a  desiccator  and  weigh  covered. 
The  loss  of  weight  is  considered  as  moisture. 

Volatile  matter.  —  Heat  one  gram  of  the  air-dry  sample  (or 
the  portion  which  has  been  used  for  the  determination  of 
moisture)  in  a  platinum  crucible  weighing  20-30  grams  with  a 
well-fitting  cover  over  the  full  flame  of  a  good  Bunsen  burner 
for  7  minutes,  in  a  place  free  from  drafts.  The  flame  should  be 
25  cm.  high  and  the  crucible  supported  on  a  platinum  triangle 
so  that  the  bottom  of  the  crucible  is  6  to  8  cm.  above  the  top 
of  the  burner  and  the  entire  crucible  is  surrounded  by  the 
hottest  part  of  the  flame.  The  upper  surface  of  the  cover 
should  burn  clean;  any  carbon  on  the  under  surface  of  the 
cover  is  weighed  with  the  residue  in  the  crucible.  The  loss 
in  weight  by  this  heating,  corrected  for  moisture  if  neces- 
sary, is  called  the  volatile  combustible,  or  simply  the  volatile 
matter. 

Ash.  —  Burn  one  gram  of  sample,  or  the  residue  from  one  of 
the  above  determinations,  in  a  platinum  crucible  (open  and  in- 
clined) first  over  a  very  low  flame,  then  at  a  higher  temperature, 
till  free  from  carbon,  and  weigh  the  residue  as  ash. 

1  Report  of  Committee  of  American  Chemical  Society  :  J.  Am.  Chem.  Soc.. 
21,  1119. 


FUELS  257 

Fixed  carbon  is  found  by  subtracting  the  sum  of  the  percent- 
ages of  moisture,  volatile  matter,  and  ash  from  100. 

The  determination  of  sulphur,  while  not  apart  of  the  proximate 
analysis,  is  so  often  required  in  connection  with  it  that  the 
usual  method  may  be  outlined  here.  This  is  the  modified 
Eschka  method.  One  gram  of  the  finely  pulverized  coal  is 
mixed  with  I'gram  of  light  magnesium  oxide  and  0.5  gram  of 
dry  sodium  carbonate  in  a  Meissen  porcelain  crucible  (or  plati- 
num dish  of  about  75  cc.  capacity)  and  heated  with  an  alcohol 
lamp  (or  with  a  gas  flame  nearly  free  from  sulphur,  the  dish 
being  set  in  a  smooth  hole  in  an  asbestos  pad  so  that  its  contents 
are  protected  as  much  as  possible  from  the  products  of  combus- 
tion). The  heat  should  be  applied  gently  at  first,  especially 
with  soft  coals,  and  the  mixture  stirred  frequently  with  a  stout 
platinum  wire.  Gradually  increase  the  heat  until  the  bottom 
of  the  dish  reaches  low  redness,  and  maintain  at  about  this 
temperature  with  frequent  stirring  until  all  carbon  is  burned; 
then  cool  and  transfer  to  a  beaker  with  about  50  cc.  water,  add 
15  cc.  bromine  water  (saturated)  and  boil  for  at  least  5  minutes 
to  complete  the  oxidation  of  the  sulphur  compounds  to  sulphates; 
allow  to  settle,  decant  the  solution  through  a  filter,  and  boil  the 
residue  a  second  and  a  third  time  with  about  30  cc.  portions  of 
water  and  then  \vash  thoroughly  with  hot  water.  Acidify  the 
filtrate  with  hydrochloric  acid  so  as  to  have  about  1  cc.  of  the 
latter  in  excess,  boil  to  expel  bromine,  and  precipitate  the  sul- 
phates by  means  of  barium  chloride,  observing  the  usual  pre- 
cautions in  obtaining,  washing,  igniting,  and  weighing  the 
barium  sulphate.  From  the  weight  of  the  latter  calculate  the 
percentage  of  sulphur  in  the  coal. 


Relation  of  Proximate   Composition  to   Calorific  Power 

The  proximate  analysis  shows  the  amount,  and  something  of 
the  character,  of  the  organic  matter  in  a  coal.  The  perform- 
ance of  such  an  analysis  requires  relatively  little  time  and  no 
special  apparatus;  the  results  thus  readily  obtained  are  un- 
doubtedly often  of  value  in  helping  to  determine  the  adapta- 


258  METHODS    OF   ORGANIC    ANALYSIS 

bility  of  a  coal  to  some  particular  purpose,  and  if  the  origin  or 
general  character  of  the  coal  is  sufficiently  well  known,  the  proxi- 
mate analysis  may  give  a  fair  indication  of  its  probable  calo- 
rific power.  Formulae  for  estimating  the  calorific  power  of 
coal  from  its  proximate  analysis  have  been  proposed,  that  of 
Goutal  being  most  often  quoted ;  but  on  the  other  hand,  it  has 
been  found  by  experiments  in  the  laboratories  of  the  United 
States  Geological  Survey,  that  the  volatile  matter  driven  off  by 
heat,  as  in  the  usual  proximate  analysis,  consists  to  a  consider- 
able degree  of  inert  gases,  and  that  the  proportion  of  these  in 
the  volatile  matter  "  varies  in  different  coal  deposits,  and  makes 
it  impossible  to  determine  the  heating  value  of  the  coal  from 
the  proximate  analysis  alone."1 

That  there  should  be  considerable  differences  in  the  calorific 
power  of  the  volatile  matter  of  different  coals  is  obvious 
when  one  considers  that  the  material  volatilized  from  some 
bituminous  coals  is  rich  in  hydrocarbons,  while  that  from 
woody  lignites  contains  a  relatively  large  amount  of  water 
vapor. 

In  order  to  obtain  definite  data  (1)  on  the  average  relation 
of  calorific  power  to  proximate  composition,  and  (2)  on  the 
extent  of  the  variations  from  the  average  relation  to  expected 
in  individual  samples  or  different  types  of  coal,  Sherman  and 
Regester  compiled  and  computed  the  results  of  analyses  made 
in  the  laboratories  of  the  United  States  Geological  Survey,  the 
Ohio  Geological  Survey,  the  West  Virginia  Geological  Survey, 
and  Columbia  University  covering  in  all  500  samples  of  coal 
from  different  parts  of  the  United  States,  and  believed  to  rep- 
resent the  principal  American  types  of  coal.  In  all  cases  the 
proximate  composition  had  been  determined  by  the  method 
outlined  above,  and  the  calorific  power  by  combustion  in  oxy- 
gen in  a  bomb  calorimeter  of  standard  type,  such  as  the 
Atwater,  Mahler,  or  Williams  instrument.  When  the  data  of 
the  500  .samples  were  calculated  to  the  basis  of  dry,  ash-free 
material,  and  grouped  according  to  percentage  of  volatile  mat- 
ter, it  was  found  that  the  coals  containing  from  2  to  10  per 
1  U.  S.  Geological  Survey,  Bui.  339,  p.  9. 


FUELS  259 

cent  of  volatile  in  the  combustible  matter  averaged  about 
14,900  B.  T.  U.  per  pound  of  combustible ;  beyond  this  the 
calorific  power  increased  with  the  volatile  matter  until  the  lat- 
ter reached  about  17  per  cent,  after  which  it  declined  gradu- 
ally with  increasing  proportion  of  volatile  in  the  combustible 
matter,  up  to  about  40  per  cent,  beyond  which  the  average 
calorific  power  decreased  more  rapidly. 

The  average  calorific  powers  for  different  percentages  of 
volatile  matter  on  the  basis  of  dry,  ash-free  coal  were  then 
plotted,  and  the  values  estimated  from  this  curve  were  com- 
pared with  those  found  by  the  bomb  calorimeter  for  each  of 
the  500  coals. 

The  first  hundred  coals  showed  from  1.74  to  8.50  per  cent  of 
volatile  matter  in  the  dry,  ash-free  substance  and  a  mean  dif- 
ference between  the  calculated  and  the  determined  values  of 
0.98  per  cent  ;  in  the  second  hundred  coals  with  8.62  to  22.17 
per  cent  volatile,  the  mean  difference  was  1.14  per  cent;  in  the 
third  hundred  with  22.53  to  38.42  per  cent  volatile,  the  mean 
difference  was  1.32  per  cent;  the  next  160  coals  showed  38.44 
to  47.93  per  cent  volatile  and  a  mean  difference  of  1.96  per  cent ; 
while  in  the  last  40  coals  which  contained  48.22  to  59.78  per 
cent  volatile  in  the  dry,  ash-free  substance  the  mean  difference 
was  6.30  per  cent. 

It  will  be  seen  from  the  latter  figure  that  among  the  coals 
having  over  48  per  cent  of  volatile  matter  in  the  dry,  ash-free 
substance  the  probable  variations  are  so  great  that  a  state- 
ment of  the  average  relation  of  proximate  analysis  to  calorific 
power  cannot  be  made  the  basis  of  any  general  rule  for  the 
estimation  of  the  latter  value  from  the  former.  For  coals  in 
this  region  of  proximate  composition  such  relations  if  used  at 
all  should  be  worked  out  for  each  particular  type  or  vein  of 
coal. 

On  the  other  hand  for  coals  containing  up  to  48  per  cent 
of  volatile  matter  in  the  dry,  ash-free  substance  the  proximate 
analysis  gives  some  indication  of  the  probable  calorific  power, 
the  average  relation  being  approximately  as  shown  in  the  follow- 
ing table. 


260 


METHODS   OF   ORGANIC   ANALYSIS 


TABLE  24.  —  APPROXIMATE  AVERAGE  RELATION  OF  VOLATILE  MATTER 
AND  CALORIFIC  POWER  IN  THE  DRY,  ASH-FREE  SUBSTANCE  OF 
AMERICAN  COALS 


Volatile  matter 
in  dry,  ash-free 
substance 
per  cent 

British  Thermal 
Units  per 
pound  of  dry, 
ash-free 
substance 

Volatile  matter 
in  dry,  ash-free 
substance 
per  cent 

British  Thermal 
Units  per 
pound  of  dry, 
ash-free 
substance 

Volatile  matter 
in  dry,  ash-free 
substance 
per  cent 

British  Thermal 
Units  per 
pound  of  dry, 
ash  -free 
substance 

0 

14544  * 

17 

15900 

33 

15500 

2 

14900      ' 

18 

15850 

34 

15500 

3 

14900 

19 

15800 

35 

15500 

4 

14900 

20 

15800 

36 

15500 

5 

14900 

21 

15700 

37 

15300 

6 

14900 

22 

15700 

38 

15100 

7 

14900 

23 

15700 

39 

15100 

8 

14900 

24 

15700 

40 

15100 

9 

14900 

25 

15700 

41 

14600 

10 

14900 

26 

15700 

42 

14600 

11 

15050 

27 

15700 

43 

14600 

12 

15200 

28 

15700 

44 

14600 

13 

15350 

29 

15700 

45 

14500 

14 

15500 

30 

15600 

46 

14400 

15 

15650 

31 

15500 

47 

14300 

16 

15800 

32 

15500 

48 

14200 

This  table  probably  gives  as  good  an  idea  as  is  now  possible 
of  the  average  relation  of  proximate  composition  to  calorific 
power  in  American  coals,  but  these  average  relations  must  not  be 
given  undue  weight,  because  individual  samples  of  coal  may  vary 
greatly  from  the  average.  An  idea  of  the  variations  to  be  ex- 
pected in  individual  cases  may  be  obtained  from  the  fact  that 
among  the  460  cases  considered  by  Sherman  and  Regester  which 
fall  within  the  range  of  this  table  the  calorific  powers  actually 
determined  by  combustion  in  oxygen  differed  from  those  calcu- 
lated from  the  above  table  by  1.00  per  cent  or  more  in  233  cases 
or  almost  exactly  one  half  of  the  total  ;  by  2.00  per  cent  or 
more  in  110  cases  (about  1  in  4)  ;  by  3.00  per  cent  or  more  in 
53  cases  (about  1  in  8)  ;  by  4.00  per  cent  or  more  in  27  cases 

1  Corresponding  to  8080  calories  per  gram,  the  usually  accepted  value  for 
carbon. 


FUELS  261 

(about  1  in  17)  ;  and  by  5.00  per  cent  or  more  in  17  cases  or 
about  1  case  in  27. 

If  this  comparison  were  extended  to  the  coals  containing  a 
larger  amount  of  volatile  matter,  the  proportion  of  cases  show- 
ing serious  discrepancies  would  of  course  be  much  greater.  It 
is  evident  that  in  general  the  proximate  analysis  of  a  coal  is  of 
less  value  as  an  indication  of  its  calorific  power  than  is  the  ulti- 
mate analysis. 

The  results  of  either  ultimate  or  proximate  analysis  become 
more  significant  when  interpreted  in  the  light  of  the  foregoing 
data;  but  in  cases  in  which  an  accurate  knowledge  of  the  calo- 
rific power  of  coal  is  required,  one  should  accept  only  the  results 
of  direct  determinations  by  a  skilled  observer  using  an  accu- 
rately standardized  bomb  calorimeter. 

REFERENCES 

I 

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II 

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262          METHODS  OF  ORGANIC  ANALYSIS 

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1906.  ABBOTT  :   Some  Characteristics   of   Coal   as   Affecting  Performance 

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1,  98. 

PARR  :   The  Classification  of  Coals.     J.  Am.  Chem.  Soc.,  28,  1425. 
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and  Lignites).     /.  Am.  Chem.  Soc.,  28,  1002,  1630. 

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26,  670. 
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1488. 
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Dept.  Agriculture,  Bureau  of  Animal  Industry,  Bui.  94. 
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FUELS  263 

1907.  HOLMES  and  RANDALL:  Testing  of  Coals  used  by  the  United  States' 

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50,  520;  Chem.  Abs.,  1,  2632. 
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Adiabatic  Method.     Z.  physik.  Chem.,  59,  532;    Chem.  Abs.,  1, 

2971. 

SY  :  Alcohol  as  a  Fuel.     J.  Frank  Inst.,  163,  57. 
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Materials,  7,  560. 
WOODWELL:  Purchase  of  Coal  under  Specifications.     Proc.  Am.  Soc. 

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1908.  JAKOB  :   (Calorimetry).     Z.  chem.  Apparatenkunde,  2,  281,  313,  337, 

369,  499,  533,  565,  597  ;  Chem  Abs.,  2,  1803. 
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NYSTROM  :  Peat  and  Lignite  ;  their  Manufacture  and  Uses  in  Europe. 

Canada  Dept.  Mines,  Ottawa,  1908,  247  pp. 
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Chem.  Abs.,  2,  650. 
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Illinois,  Engineering  Experiment  Station,  Bui.  17,  p.  26. 
PORTER  and  OVITZ  :  The  Nature  of  the  Volatile  Matter  of  Coal  as 

Evolved  under   Different  Conditions.     J.  Am.   Chem.  Soc.,  30, 

1486. 
REDWOOD  :  Supply  and  Use  of  Mineral  Oil.     Engineering,  86,  118 ; 

Chem.  Abs.,  3,  372. 

WOODWELL  :  Commercial  Results  in  the  Purchase  of  Coal  on  Specifi- 
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1909.  BAILEY  :    Accuracy  in  Sampling  Coal.     /.  Ind.  Eng.  Chem.,  1,  161. 

:  Calorimeter  Standardization.     J.  Ind.  Eng.  Chem.,  1,  328. 

BRINSMAID  :  Amount  of  Inert  Volatile  Matter  in  the  Mineral  Con- 
stituents of  Coal.     J.  Ind.  Eng.  Chem.,  1,  65. 

Cox  :  Coal  Calorimetry.     Philippine  J.  Sci.  (A),  4,  171;  Chem.  Abs., 

3,  3006. 

EMERSON  :  A  New  Bomb  Calorimeter.     /.  Ind.  Eng.  Chem.,  1,  17. 
FISCHER  and  WREDE  :  (Use  of  Platinum  Resistance  Thermometer  in 

Determining  Heat  of  Combustion).     Z.  physik.  Chem.,  69,  218  ; 

Chem.  Abs.,  4,  537. 
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Agriculture,  Bureau  of  Animal  Industry,  Bui.  124. 
LANGBEIN  :  Modified  Bomb  Calorimeter.     Chem.  Ztg.,  33,  1055. 


264  METHODS    OF   ORGANIC    ANALYSIS 

1909.  LORD  :  Coal  Analysis.     /.  Ind.  Eng.  Chem.,  1,  307. 

PARR  et  al :  Determination  of  Sulphur  in  Coal.     J.  Ind  Eng.  Chem., 

1,  689. 
PARR  and  WHEELER  :  The  Ash  of  Coal  and  its  Relation  to  Actual  or 

Unit  Coal  Value.     J.  Ind.  Eng.  Chem.,  1,  636. 
SHIMER  :   The  Determination  of  the  Volatile  Combustible  Matter  in 

Coke  and  Anthracite.     J.  Ind.  Eng.  Chem.,  1,  518. 

1910.  BEMENT  :    Influence  of  Oxygen  on  the  Value  of  Coal.     Science,  30,  922. 
BENEDICT  and  HIGGINS  :   An  Adiabatic  Calorimeter  for  Use  with  the 

Calorimetric  Bomb.     J.  Am.  Chem.  Soc.,  32,  461. 
BURGESS  and  WHEELER:   Volatile  Constituents  of  Coal.     J.  Chem. 

Soc.,  97,  1917. 
FIELDNER  and  DAVIS  :    Some  Variations  in  the  Official  Method  for 

the  Determination  of  Volatile  Matter  in  Coal.      J.  Ind.  Eng. 

Chem.,  2,  304. 
HUNTLEY  :   Accuracy  Obtainable  in  Fuel  Calorimetry.     J.  Soc.  Chem. 

Ind.,  29,  917. 
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116. 

PARR  :   A  New  Gas  Calorimeter.     J.  Ind.  Eng.  Chem.,  2,  337. 
PARR  and  BARKER  :  Occluded  Gases  in  Coal.     Univ.  of  III.  Bui.,  6,  32. 
PARR  and  WHEELER  :   Unit  Coal  and  the  Composition  of  Coal  Ash. 

Univ.  of  III.  Bui,  6,  43 ;  Chem.  Abs.,  4,  2199. 
PORTER  and  OVITZ  :   Losses  in  the  Storage  of  Coal.     /.  Ind.  Eng. 

Chem.,  2,  77. 

-  :    Volatile  Matter  in  Coal.     U.  S.  Bur.  Mines,  Bui.  1. 
RICHARDS  and  JESSE  :    Heats  of   Combustion  of  the  Octanes  and 

Xylenes.     Jf  Am.  Chem.  Soc.,  32,  268. 
WALSH:    Standard  Gas  Coal.     Progressive  Age,  28,  328;  Chem.  Abs., 

4,  2724. 
WELD:   Accuracy  in  Sampling  Coal.     J.  Ind.  Eng.  Chem.,  2,  426. 

(See  also  p.  543.) 

WHITE  :    (Temperature  Measurements  in  Calorimeter  Work).     Physi- 
cal Review,  31,  562. 

1911.  ALLEN  :   Specifications  for  the  Purchase  of  Fuel  Oil  for  the  Govern- 

ment with  Directions  for  Sampling  Oil  and  Natural  Gas.     J.  Ind. 

Eng.  Chem.,  3,  730. 
ANONYMOUS  :   Estimation  of  Moisture  in  Fuel  Oil.     Chem.  Tech.  Ztg., 

1911  (6),  29,  47;  Chem.  Abs.,  5,  2425. 
BLAKELEY   and   CHANCE  :   Accurate   Technical  Estimation   of   the 

Calorific  Power  of  Anthracite  Coal.     J.  Ind.  Eng.  Chem.,  3,  557. 
BURGESS    and    WHEELER:    The    Volatile    Constituents    of    Coal. 

/.  Chem.  Soc.,  99,  649. 
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FUELS  265 

1911.   DOANE  :   The  Purchase  of  Coal  on  the  Efficiency  Basis.     Eng.  Rec., 

61,  502;  Chem.  Abs.,  5,  584. 
HOLMES  :    The  Sampling  of  Coal  in  the  Mine.     Technical  Paper  1, 

U.  S.  Bur.  Mines. 
KENT  and  ALLEN  :  Formula  for  Purchase  of  Coal  on  Heat  Unit  Basis. 

Eng.  News,  65,  109. 
PARKER  :   The  Production  of  Coal  in  1910.     Published  by  U.  S.  Geol. 

Survey.     (This  includes  a  classified  list  (pp.  226-241)   of  the 

papers  dealing  with  coal,  coke,  lignite,  and  peat  contained  in 

publications  of  the  U.  S.  Geological  Survey.) 
PARR  :   The  Determination  of  Volatile  Matter  in  Coal.     /.  Ind.  Eng. 

Chem.,  3,  900. 
POPE  :   Purchase  of  Coal  by  the  Government  under  Specifications. 

U.  S.  Geol.  Survey,  Bur.  Mines,  Bui.  11. 
U.  S.  Bureau  of  Standards.     Circular  No.  11,  The  Standardization  of 

Bomb  Calorimeters. 
WHITE  :   Recent  Progress  in  Calorimetry.     Met.  Chem.  Eng.,  9,  202, 

296,  449. 


CHAPTER   XIII 


Soap  and  Glycerin 

ANALYSIS  OF   COMMERCIAL   SOAP 

THE  determinations  required  in  the  examination  of  a  com- 
mercial soap  depend  largely  upon  the  purpose  for  which  it  is 
intended.  The  scheme  outlined  below  is  adaptable  to  almost 
all  cases,  as  it  can  be  easily  extended  to  include  additional  deter- 
minations or  shortened  by  omitting  such  steps  as  are  unneces- 
sary when  only  a  partial  analysis  is  required. 

OUTLINE  SCHEME  OF  ANALYSIS1 

FIRST  PORTION.  Dry  2  to  5  grams  of  soap  (I)2  and  introduce  into 
Soxhlet  extractor,  either  in  a  thimble  or  supported  by  a  firm  plug  of  cotton, 
and  extract  with  petroleum  ether. 


Solution.     Evap- 
orate    ether     and 
weigh  as  unsaponi- 
Jied  fat  and  unsa- 
ponifidble     matter. 
(II.) 

Residue.     Allow  petroleum  ether  to  evaporate,  trans- 
fer the  soap  to  a  beaker,  dissolve  in  hot  water,  and  filter. 

Solution.     Decompose  with  a  con- 
siderable excess   of    standard    sul- 
phuric (or  hydrochloric)  acid,  sep- 
arate aqueous  solution  from  fatty 
layer.     (III.) 

Residue.     Dry 
and   weigh  as   in- 
soluble matter. 
Ignite  and  weigh 
as  insoluble  mineral 
matter.     (X.) 

Solution.     Add 
methyl    orange 
and    titrate    for 
total  alkali.     Test 
for  chlorides  (or 
sulphates),  sugar, 
and     glycerin. 
(IV.) 

Fatty   Layer. 
Dry  and  weigh  as 
fatty   and    resin 
acids.     (V.) 

1  Allen :  Commercial  Organic  Analysis,  Vol.  II. 

2  Numbers  in  parenthesis  refer  to  sections  which  follow. 

266 


SOAP    AND    GLYCERIN 


267 


SECOND  PORTION.     Exhaust  2  to  5  grams  of  the  fresh  sample  with  care- 
fully neutralized  alcohol.     (VI.) 


Solution.      Add 
pheuolphthalein 
and  titrate  for  free 
caustic  alkali   or 
free  fatty  acid. 
(VII.) 

Residue.      Dry    and    weigh.       (VIII.)      Exhaust 
thoroughly  with  boiling  water. 

Solution.       Divide     into    aliquot 
parts. 

Residue.    May  be 
examined    instead 
of     residue     from 
first     portion     or 
used  for  additional 
tests  or  determina- 
tions.    (X.) 

Add    methyl 
orange  and  ti- 
trate    for    alkali 
carbonate  (and 
b  or  ate,  silicate, 
or    aluminate   if 
present).     (IX.) 

Test   for  (and 
if    necessary   de- 
termine) sulphate, 
borate,    silicate, 
and  aluminate. 
(IX.) 

A  cake  of  soap  exposed  to  the  air  dries  rapidly  at  the  surface, 
forming  a  horny  layer  which  to  some  extent  prevents  the  evap- 
oration of  water  from  the  interior.  In  sampling  such  a  soap  it 
is  important  to  remember  this  variation  in  water  content  of 
different  parts  of  the  same  cake.  If  the  sample  is  to  represent 
the  individual  cake  as  it  existed  at  the  time  of  commencing  the 
analysis,  a  number  of  sections  through  the  entire  cake  should 
be  taken  either  by  slicing  or  by  means  of  a  cork  borer  or  a 
cheese  or  butter  sampler.  Often  the  purpose  of  the  analysis  is 
to  show  the  composition  of  the  sample  as  originally  sold,  in 
which  case  the  dried  surface  should  be  rejected  and  the  sample 
taken  from  the  center  of  the  cake.  The  sample  for  analysis 
should  be  reduced  quickly  to  fine  shavings  and  kept  in  a  tightly 
stoppered  bottle. 

DETAILS  OF  DETERMINATIONS   INDICATED   IN  THE  SCHEME 

I.    Determination  of  Water 

If  the  soap  is  very  hard  and  dry,  it  may  be  reduced  to  fine 
shavings  and  dried  on  a  watchglass,  heating  first  for  some  time 
at  40°  to  60°  and  then  finishing  at  105°  to  110°.  Very  few 
soaps  can  be  completely  freed  from  moisture  in  this  way,  but 
some  others  may  be  sufficiently  dried  for  the  ether  extraction, 
while  the  moisture  determination  is  made  on  a  separate  sample 


268  METHODS    OF    ORGANIC   ANALYSIS 

as  follows :  Dissolve  about  two  grams  of  soap  in  the  minimum 
quantity  of  hot  strong  alcohol  and  evaporate  on  clean  dry  sand. 
Finish  the  drying  at  110°  with  frequent  stirring,  a  small  rod 
having  been  weighed  with  the  dish.  For  approximate  deter- 
mination of  moisture  Allen  recommends  Smith's  method:  Heat 
5  to  10  grams  of  finely  divided  soap  in  a  large  porcelain  crucible 
on  a  sand  bath  over  a  small  Bunseu  flame.  Stir  continually 
.with  a  small  glass  rod  (weighed  with  the  crucible)  having  a 
roughened  end  to  facilitate  breaking  any  lumps  of  soap  which 
may  be  formed.  Continue  heating  until  there  is  no  more  evi- 
dence of  water  being  expelled,  then  test  by  removing  the  burner 
and  placing  a  cold  glass  at  once  over  the  crucible.  If  no  moist- 
ure condenses  on  the  glass,  the  soap  is  considered  dry.  With 
practice  the  drying  can  be  finished  in  20  to  30  minutes.  Any 
burning  of  the  soap  is  readily  detected  by  the  odor.  The 
results  are  said  to  be  reliable  to  0.25  per  cent. 

II.    Petroleum  Ether  Extract 

Transfer  the  thoroughly  dried  soap  to  a  paper  "  fat  extraction 
thimble,"  plug  tightly  with  fat-free  cotton  and  treat  in  a  .Soxh- 
let  extractor,1  heated  on  a  safety  water  bath  or  electric  heater, 
with  petroleum  ether  of  nearly  constant  boiling  point.  Regu- 
late the  heating  so  that  the  extractor  fills  to  the  siphoning  point 
in  ten  or  fifteen  minutes  and  continue  the  extraction  for  four 
to  six  hours.  Disconnect  the  extraction  apparatus  (observing 
that  no  free  flame  is  near)  as  the  solvent  flows  through  the 
siphon  to  the  flask;  remove  the  thimble,  reconnect  the  appara- 
tus, and  recover  the  solvent  by  placing  a  wide,  short  test-tube  in 
the  space  previously  occupied  by  the  thimble,  or  by  allowing 
the  solvent  to  collect  in  this  space  and  removing  it  before  it 
reaches  the  top  of  the  siphon.  Having  expelled  nearly  all  of 
the  petroleum  ether,  heat  the  flask  containing  the  extract  in  a 
boiling  water  oven  to  constant  weight. 

The  petroleum  ether  extract  of  a  commercially  pure  soap 

1  The  parts  of  the  Soxhlet  extractor  may  be  connected  by  ground  glass  joints, 
by  mercury  seal,  or  by  corks  covered  with  tinfoil. 


SOAP   AND   GLYCERIN  269 

may  contain  unsaponified  fat  (or  free  fatty  acids,  which  are 
now  largely  used  in  soap-making)  as  well  as  the  "  unsaponifiable 
matter  "  of  the  original  soap  grease.  Hydrocarbons,  phenols, 
and  other  substances  soluble  in  petroleum  ether  may  also  be 
found  in  mixed  and  "  medicated  "  soaps.  Allen l  gives  direc- 
tions for  the  systematic  examination  of  this  extract,  including 
the  quantitative  determination  of  phenol  if  present. 

III.  Liberation  of  Fatty  and  Resin  Acids 

Decompose  the  water  solution  of  the  soap  by  adding  a  consid- 
erable excess  (allow  5  to  7  cc.  of  normal  acid  for  each  gram  of 
dry  soap)  of  normal  or  half-normal  sulphuric,  nitric,  or  hydro- 
chloric acid.  Add  the  entire  amount  of  acid  at  once,  boil,  stir 
thoroughly  for  some  minutes,  and  keep  the  solution  hot  until 
the  fatty  acids  collect  at  the  surface,  leaving  the  water  solution 
nearly  clear,  then  complete  the  separation  as  in  the  determina- 
tion of  insoluble  acids  in  butter. 

It  is  convenient  to  liberate  the  fatty  acids  in  a  tared  beaker 
in  which  they  can  afterward  be  dried  and  weighed  (V).  If 
the  fatty  acids  are  liquid  at  ordinary  temperature  or  form  a 
cake  too  soft  to  be  handled  conveniently,  a  known  weight  of 
dry  bleached  beeswax  or  stearic  acid  may  be  added  to  the  hot 
solution.  The  fatty  acids  become  incorporated  with  the  wax 
and  on  cooling  a  firm  cake  is  obtained. 

IV.  Solution  separated  from  Fatty  Acids 

This  solution  contains,  in  the  form  of  sulphate  (or  chloride  if 
hydrochloric  acid  be  used  to  decompose  the  soap),  all  the  alkali 
originally  present  as  soap,  as  carbonate  (silicate  or  borate),  or 
as  hydroxide.  On  titrating  this  solution  with  alkali,  using 
methyl  orange  as  indicator,  the  amount  of  acid  found  to  have 
been  neutralized  gives  a  measure  of  the  total  alkali  of  the  soap.2 

1  Commercial  Organic  Analysis,  Vol.  II. 

2  A  quick  determination  of  total  alkali  can  also  be  made  by  burning  a  weighed 
portion  of  the  soap  to  a  white  ash  and  determining  the  alkalinity  of  this  ash,  using 
methyl  orange  as  indicator. 


270  METHODS   OF   ORGANIC   ANALYSIS 

Unless  potash  is  known  to  be  present,  this  total  alkali  is  usually 
calculated  as  sodium  oxide.  The  solution  also  contains  any  chlo- 
rides or  other  soluble  salts,  soluble  fatty  acids,  glycerol,  sugar, 
etc.,  which  the  soap  may  have  contained.  After  titration  the 
solution  can  be  diluted  to  a  known  volume  and  separate  por- 
tions taken  for  qualitative  tests  and  quantitative  determinations. 

When  sulphuric  acid  has  been  used  to  liberate  the  fatty  acids, 
chlorides  can  be  determined  in  a  portion  of  the  neutralized  solu- 
tion. If  it  is  desired  to  determine  both  chloride  and  sulphate 
in  this  solution,  the  soap  can  be  decomposed  by  means  of  stand- 
ard nitric  acid.  It  is  usually  more  convenient  to  test  for  sul- 
phates in  the  residue  from  the  alcohol  extraction  as  described 
below. 

Soluble  fatty  acids  will  be  found  in  this  solution  if  the  liberated 
acids  of  coconut  or  palm-nut  oil  soaps  are  washed  with  hot 
water,  as  is  often  recommended.  When  the  fatty  acids  are 
separated  cold  and  washed  with  cold  water  only,  the  amount 
dissolved  can  usually  be  neglected  without  appreciable  error. 

Sugar,  if  present,  is  detected  in  a  part  of  this  solution.  After 
further  treatment  with  acid  to  insure  complete  hydrolysis,  the 
invert  sugar  is  determined  either  volumetrically  or  gravimetri- 
cally  (Chapter  III).  Sugar  may  also  be  determined  by  means 
of  the  polariscope,  using  a  separate  portion  of  the  sample  and 
precipitating  the  fatty  acids  as  insoluble  barium  soaps.1 

In  the  absence  of  sugar  and  other  interfering  substances, 
glycerol  can  be  determined  by  treating  a  portion  of  the  neu- 
tralized solution  directly  with  sulphuric  acid  and  standard 
dichromate  as  described  beyond.  Since  the  results  thus  found 
are  often  too  high,  because  of  the  presence  of  organic  impurities, 
Lewkowitsch  recommends  the  following  method:  Decompose 
the  water  solution  of  the  soap  with  sulphuric  acid,  separate  the 
fatty  acids,  neutralize  the  nitrate  with  barium  carbonate,  evapo- 
rate to  a  sirup,  and  extract  with  a  mixture  of  3  parts  95  per 
cent  alcohol  and  1  part  ether.  The  glycerol  thus  obtained  can 
be  determined  by  the  acetin  method  after  complete  removal  of 
alcohol. 

iFreyer:  Oesterr.  Chem.  Ztg.,  1900,  3,  25;  Analyst,  1900,  25,  127. 


SOAP   AND   GLYCERIN  271 

V.    Mixed  Fatty  and  Resin  Acids 

The  mixture  of  acids  liberated  as  already  described  (III)  is 
dried  to  constant  weight  in  a  boiling  water  oven  in  a  weighed 
flat-bottomed  dish  or  beaker  as  in  the   determination  of  the 
insoluble  acids  in  butter  fat.     The  weight  having  been  found, 
test  a  portion  for  resin  acid  by  the  Liebermann-Storch  reaction 
as  described  under  drying  oils  (Chapter  X).     In  the  absence  of 
resin  acids  it  may  be  possible  to  show  the  nature  of  the  fat  from 
which  the  soap  was  made,  by  examining  the  mixed  fatty  acids 
according  to  the  methods  used  in  identifying  fats  and  oils  as 
described   in    Chapters  VIII  to  XI,  consulting  special  works 
such  as  those  of  Lewkowitsch  or  Benedikt-Ulzer  for  the  "con- 
stants "  which  cannot  be  inferred  from  the  properties  of  the  cor- 
responding fats.      The  separation  of  fatty  and  resin  acids  is  best 
accomplished  by  TwitcheWs  method  based  upon  the  difference  of 
behavior  of  these  acids  when  exposed  in  alcoholic  solution  to 
the  action  of  hydrochloric  acid.     By  this  treatment  fatty  acids 
are  converted  to  ethyl  esters,  while  resin  acids  remain  practically 
unchanged.     The  method  is  carried  out  by  Lewkowitsch  l  as 
follows:     Weigh  2  to  3  grams  of  the  mixed  acids  in  a  flask, 
dissolve  in  10  times  their  volume  of  absolute  alcohol,  immerse 
the  flask  in  cold  water,  and  pass  a  current  of  dry  hydrochloric 
acid  gas  through  the  solution  for  an  hour;  then  dilute  the  con- 
tents of  the  flask  (which  will  have  separated  into  two  layers) 
with  5  times  its  volume  of  water  and  boil  until  the  aqueous 
solution  has  become  clear,  the  esters,  with  resin  acids  in  solu- 
tion, floating  on  top.     Transfer  the  contents  of  the  flask  to  a 
separating  funnel  by  means  of  50  cc.  of  petroleum  ether  (boil- 
ing below  80°);    shake,  allow  to  separate,  draw  off  the  acid 
solution,  and  wash  the  petroleum  ether  layer  once  with  water. 
After  the  latter  has  separated  completely  and  been  removed, 
add  a  solution  of  0.5  gram  of  potassium  hydroxide  and  5  cc. 
of  alcohol  in  50  cc.  of  water;   shake,  and  allow  to  separate. 
The  ethyl  esters  remain  dissolved  in  the  petroleum  ether,  while 
the  resin  acids  are  extracted  by  the  dilute  alkaline  solution 
1  Oils,  Fats,  and  Waxes  (4th  Ed.),  p.  501. 


272  METHODS   OF   ORGANIC    ANALYSIS 

forming  soaps.  Draw  off  the  soap  solution,  wash  the  petro- 
leum ether  solution  again  with  dilute  alkali,  unite  the  alkaline 
solutions;  liberate  the  resin  acids  by  means  of  hydrochloric 
acid,  collect,  dry,  and  weigh  them  as  in  the  determination  of 
liberated  fatty  acids. 

The  resin  acids  can  be  titrated  after  washing  free  from 
hydrochloric  acid,  instead  of  being  separated  and  weighed. 
The  volumetric  method  is  more  rapid  than  the  gravimetric,  but 
necessitates  the  assumption  of  a  combining  weight  (346)  for 
the  resin  acids,  which  is  liable  to  considerable  inaccuracy. 
According  to  Lewkowitsch,  the  results  by  the  volumetric 
method  are  likely  to  be  too  high  ;  those  by  the  gravimetric 
method  too  low. 

VI.    Extraction  with  Alcohol 

Dry  soap  can  be  extracted  with  95  per  cent  alcohol  ;  for 
wet  soap  stronger  alcohol  should  be  used,  so  that  after  taking 
up  the  moisture  of  the  sample  it  will  still  be  too  strong  to  dis- 
solve an  appreciable  amount  of  carbonate.  The  alcohol  to  be 
used  must  first  be  very  carefully  neutralized,  using  phenol- 
phthalein  as  indicator.  In  this  neutralization  there  is  danger 
of  adding  an  excess  of  alkali  unless  it  is  remembered  that  the 
full  pink  color  of  the  indicator  will  not  appear  in  alcohol  of 
this  strength.  If  difficulty  is  experienced  in  detecting  the 
neutral  point,  a  small  amount  of  the  alcohol  can  be  removed 
and  mixed  with  an  equal  volume  of  boiling  water  to  bring  out 
the  color  of  the  indicator. 

While  the  extraction  of  the  soap  with  alcohol  is  often  carried 
out  in  open  vessels,  filtering  and  washing  in  the  ordinary  way, 
it  is  usually  more  satisfactory  to  use  the  Soxhlet  extractor. 
The  soap  can  be  put  in  a  paper  thimble  as  in  the  petroleum 
ether  extraction,  or  between  plugs  of  cotton  in  a  glass  tube 
with  perforated  bottom.  In  the  latter  case  the  progress  of 
the  extraction  can  be  watched  without  disconnecting  the 
apparatus.  When  the  extraction  is  complete,  the  residue 
should  be  in  powder  form.  If  distinct  pieces  remain,  these 
may  contain  soap  which  has  been  protected  from  the  action 


SOAP   AND    GLYCERIN  273 

of  the  alcohol  by  the  formation  of  a  layer  of  insoluble  salts. 
In  this  case  remove  and  crush  the  residue,  replace,  and 
extract  again. 

VII.   Free   Caustic  Alkali  or  Fatty  Acid 

To  the  alcoholic  extract  add  a  few  drops  of  neutralized  phe- 
nolphthalein  solution.  If  the  solution  reacts  alkaline,  titrate 
with  tenth-normal  acid  for  caustic  alkali;  if  acid,  titrate  with 
tenth-normal  alkali  for  free  acid.  For  a  further  discussion  of 
this  extract,  including  a  rapid  method  for  the  partial  analysis 
of  soaps,  see  Allen. 

VIII.    Residue  Insoluble  in  Alcohol 

It  is  advisable  to  dry  and  weigh  this  residue  so  that  the  per- 
centage of  impurities  not  actually  determined  can  be  found  by 
difference.  A  microscopic  examination  may  also  be  of  use  in 
determining  the  subsequent  treatment.  Starch  and  gelatin  if 
present  could  be  separated  from  carbonate,  borate,  and  sulphate 
by  dissolving  the  latter  salts  in  cold  water  ;  but  silicate  would 
probably  be  incompletely  dissolved,  and  it  is  therefore  better 
as  a  rule  to  extract  with  hot  water  and  to  use  separate  portions 
of  the  soap,  if  necessary,  for  the  determination  of  starch  and 
gelatin.  In  such  a  case  extract  the  soap  with  alcohol  and  in 
the  residue  determine  starch  as  described  in  Chapter  V,  or 
determine  nitrogen  by  the  Kjeldahl  method  and  calculate  the 
corresponding  amount  of  gelatin,  taking  the  nitrogen  content 
of  the  latter  as  17.9  per  cent.1 

IX.    Carbonate,  Silicate,  Borate,  and  Aluminate 

Add  to  the  water  extract  from  the  residue  insoluble  in 
alcohol  an  excess  of  standard  acid  and.  boil  to  insure  decom- 
position of  the  silicate.  If  it  is  important  to  distinguish  quan- 
titatively between  carbonate  and  the  other  alkaline  salts 
present,  the  carbonic  acid  given  off  during  this  boiling  can  be 
collected  and  weighed.  Add  methyl  orange  as  indicator  and 
1  Richards  and  Gies :  Am.  J.  PhysioL,  1902,  7,  129. 


274  METHODS   OF   ORGANIC    ANALYSIS 

titrate  with  standard  alkali  to  determine  the  total  amount  of 
alkali  which  was  present  as  carbonate,  silicate,  borate,  and 
aluminate. 

To  a  portion  of  the  solution  add  hydrochloric  acid  in  excess, 
evaporate  to  small  volume,  and  test  for  boric  acid  by  means  of 
turmeric  paper  ;  when  dry,  heat  at  110°,  take  up  with  dilute 
hydrochloric  acid,  filter  out,  and  determine  silica,  if  present. 

Other  portions  of  the  solution  or  the  filtrate  from  silica  can 
be  used  for  the  detection  and  determination  of  sulphates,  alu- 
rninates,  etc. 

X.  Insoluble  Matter 

This  residue  should  be  dried  to  constant  weight  at  100°,  a 
portion  examined  microscopically  and  the  remainder  ignited  and 
weighed.  If  over  1  per  cent  of  insoluble  mineral  matter  is 
found,  it  should  be  analyzed.  Among  the  substances  which 
may  be  found  in  this  residue  are  oatmeal,  bran,  sawdust,  clay, 
chalk,  steatite,  infusorial  earth,  pumice,  sand,  mineral  pigments, 
etc. 

CALCULATION  AND  INTERPRETATION  OF  RESULTS 

In  the  case  of  hard  soap,  the  results  of  the  partial  analysis 
usually  required  may  be  reported  as  follows  : 

Water  ;  unsaponified  fat  and  unsaponifiable  matter  ;  fatty 
and  resin  anhydrides  (97  per  cent  of  the  weight  of  free  acids); 
sodium  oxide  combined  as  soap  ;  sodium  hydroxide  ;  sodium 
carbonate  ;  insoluble  organic  matter  ;  insoluble  mineral  matter. 
It  is  well  to  report  also  the  total  alkali  in  terms  of  sodium 
oxide. 

The  purpose  for  which  a  soap  is  intended  must  be  known  be- 
fore an  opinion  as  to  its  quality  can  safely  be  formed.  In  most 
cases  the  percentage  of  alkali  combined  as  soap  is  the  best 
measure  of  the  amount  of  actual  soap  in  the  material,  but  for 
special  purposes  the  presence  or  absence  of  other  constituents 
is  often  of  greater  importance. 

Toilet  soaps  should  contain  as  little  free  alkali  (either  caustic 
or  carbonate)  as  possible.  Alder-Wright  divided  toilet  soaps 
into  three  classes  according  to  the  proportion  of  free  alkali  to 


SOAP   AND    GLYCERIN  275 

alkali  combined  as  soap.  The  first  class  included  those  soaps 
which  contained  less  than  2.5  per  cent  as  much  free  as  com- 
bined alkali ;  the  second,  those  in  which  the  percentage  was 
2.5  to  7.5  ;  the  third,  those  containing  over  7.5  per  cent  as 
much  free  as  combined  alkali.  In  judging  the  quality  of  toilet 
soaps  it  is  also  important  to  consider  the  proportions  and  nature 
of  all  foreign  matter,  the  amount  of  water,  the  hardness  of  the 
soap,  and  in  some  cases  the  origin  must  be  sought  by  an  exami- 
nation of  the  fatty  acids.  The  more  expensive  "  transparent " 
toilet  soaps  may  contain  alcohol  or  glycerin  ;  in  cheaper  grades 
a  similar  appearance  is  obtained  by  the  addition  of  sugar. 

Household  soaps  are  made  from  cheaper  and  softer  fats  than 
those  used  for  toilet  soap.  Alkali  in  the  form  of  carbonate, 
silicate,  or  borate  is  not  objectionable  unless  present  in  exces- 
sive amount.  No  appreciable  amount  of  sugar  or  glycerol  is 
likely  to  be  present.  Scouring  soaps  often  contain  large 
amounts  of  pulverized  quartz,  infusorial  earth,  etc.,  and  are 
sometimes  strongly  alkaline  with  sodium  carbonate  or  hydrox- 
ide. 

For  discussion  of  the  adaptability  of  different  types  of  soaps 
to  specific  uses  see  references  at  the  end  of  the  chapter. 

GLYCEROL 

Glycerol  is  a  colorless,  odorless,  viscous  liquid  of  sweet  taste 
and  neutral  reaction,  miscible  in  all  proportions  with  water 
and  with  alcohol.  It  also  dissolves  in  mixtures  of  alcohol  and 
ether,  but  is  only  very  sparingly  soluble  in  pure  ether  1  and  is 
practically  insoluble  in  chloroform,  carbon  disulphide,  and 
benzene.  The  specific  gravity  of  pure  glycerol  at  15°  referred 
to  water  at  the  same  temperature  is  variously  stated  at  from 
1.265  to  1.2677.  Anhydrous  glycerol  boils  at  about  290°,  but 
evaporates  rapidly  at  lower  temperatures  (160°  or  over),  and 
the  evaporation  is  greatly  accelerated  by  the  presence  of  a  small 
amount  of  water.  When  kindled,  glycerol  burns  with  a  blue 
flame  and  leaves  no  carbonaceous  residue. 

1  According  to  Lewkowitsch,  one  part  of  glycerol  of  1.23  sp.  gr.  dissolves  in 
about  500  parts  of  ether. 


276  METHODS  OF  ORGANIC  ANALYSIS 

These  properties,  together  with  the  fact  that  it  yields  acrolein 
when  heated  with  acid  potassium  sulphate,  are  usually  sufficient 
for  the  identification  of  glycerol  when  in  a  fairly  pure  and  con- 
centrated state. 

Glycerol  is  a  good  solvent  for  many  substances,  both  organic 
and  inorganic,  and  its  presence  often  increases  their  solubility  in 
aqueous  and  alcoholic  solutions.  This  fact  and  the  difficulty  of 
distilling  without  loss  make  it  troublesome  to  separate  glycerol  as 
a  pure  aqueous  solution  as  is  done  in  the  determination  of  alcohol. 

The  percentage  of  glycerol  in  commercial  glycerin  is  usually 
determined  either  by  acetylating  the  glycerol  and  finding  the 
amount  of  acetin  by  saponification  (acetin  method),  or  by 
quantitative  oxidation  of  the  glycerol  by  means  of  standard 
potassium  dichromate  (dichromate  method).  The  acetin 
method  requires  that  the  glycerol  be  concentrated,  and  that 
other  acetylizable  substances  if  present  shall  be  corrected  for ; 
the  dichromate  method  requires  the  removal  of  chlorides  and 
all  organic  substances  which  would  be  oxidized  by  the  dichro- 
mate treatment. 

ANALYSIS  OF  CRUDE  GLYCERIN 

In  recent  years  the  increase  in  price  of  crude  glycerin  has 
resulted  in  the  manufacture  of  glycerin  from  lower-grade  fats 
than  before,  with  the  result  that  the  product  often  contains 
impurities  which  behave  so  much  like  glycerol  as  to  introduce 
serious  discrepancies  in  the  analytical  determination  of  glycerol 
in  crude  glycerin.  This  led  to  the  appointment  of  committees, 
both  in  this  country  and  in  Europe,  to  study  methods  of  glyc- 
erin analysis.  Representatives  of  these  committees  met  as  an 
international  committee,  which,  after  investigation,  decided 
that  the  acetin  method  should  be  the  basis  on  which  glycerin 
should  be  bought  and  sold,  but  that  the  dichromate  method,  be- 
ing more  convenient  for  factory  control,  might  continue  to  be 
used  for  some  technical  purposes  in  a  properly  standardized 
form.  The  methods  recommended  by  the  international  com- 
mittee for  sampling  and  analysis  of  glycerin  are  as  follows :  1 
*J.  Ind.  Eng.  Chem.,  3,  679-686. 


SOAP   AND    GLYCEKIN  277 

Sampling 

The  most  satisfactory  method  available  for  sampling  crude 
glycerin  liable  to  contain  suspended  matter,  or  which  is  liable 
to  deposit  salt  on  settling,  is  to  have  the  glycerin  sampled  by  a 
sampler  mutually  approved  by  the  buyer  and  seller  as  soon  as 
possible  after  the  glycerin  is  filled  into  drums,  but  in  any  case 
before  any  separation  of  salt  has  taken  place.  In  such  cases  he 
shall  sample  with  a  sectional  sampler  (see  appendix  to  original 
report),  then  seal  the  drums,  brand  them  with  a  number  for 
identification,  and  keep  a  record  of  the  brand  number.  The 
presence  of  any  visible  salt  or  other  suspended  matter  is  to  be 
noted  by  the  sampler,  and  a  report  of  the  same  made  in  his  cer- 
tificate, together  with  the  temperature  of  the  glycerin.  Each 
drum  must  be  sampled.  Glycerin  which  has  deposited  salt  or 
other  solid  matter  cannot  be  accurately  sampled  from  the  drums, 
but  an  approximate  sample  can  be  obtained  by  means  of  a  sec- 
tional sampler  which  will  allow  a  complete  vertical  section  of 
the  glycerin  to  be  taken,  including  any  deposit. 

Analysis 

1.  Determination  of  Free  Caustic  Alkali.  —  Put  20  grams  of 
the  sample  into  a  100-cc.  flask,  dilute  with  approximately  50  cc. 
of  freshly  boiled  distilled  water,  add  an  excess  of  neutral  barium 
chloride  solution,  1  cc.  of  phenolphthalein  solution,  make  up  to 
the  mark,  and  mix.     Allow  the  precipitate  to  settle,  draw  off 
50  cc.  of  the  clear  liquid,  and  titrate  with  normal  acid.     Calcu- 
late the  percentage  of  caustic  alkali  as  Na2O. 

2.  Determination  of  Ash  and  Total  Alkalinity.  —  Weigh  2-5 
grams  of  the  sample  in  a  platinum  dish,  burn  off  the  glycerin 
over  a  luminous  Argand  burner  or  other  source  of  heat  at  a  low 
temperature,  to  avoid  volatilization  and  the  formation  of  sul- 
phides.    When  the  mass  is  thoroughly  charred,  stir  with  hot 
water,  filter,  wash,  and  ignite  the  residue  in  the  platinum  dish. 
Return  the  filtrate  and  washings   to  the  dish,  evaporate  the 
water,  ignite  carefully,  avoiding  fusion,  and  weigh  the  ash.     Dis- 
solve the  ash  in  water  and  titrate  total  alkalinity,  using  as  indi- 


278  METHODS  OF  ORGANIC  ANALYSIS 

cator  methyl  orange  in  a  cold  solution,  or  litmus,  if  the  solution 
is  boiled. 

3.  Determination  of  Alkali  present  as   Carbonate.  —  Take  10 
grams  of  the  sample,  dilute  with  50  cc.  water,  add  sufficient 
normal  acid  to  neutralize  the  total  alkali  found  at  (2),  boil 
under  a  reflux  condenser  for  15—20  minutes,  wash  down  the 
condenser  tube  with  water  free  from  carbon  dioxide,  and  then 
titrate  the  free  acid  in  the  solution  with  normal  sodium  hydrox- 
ide, using  phenolphthalein  as  indicator.     Calculate  the  percent- 
age of  Na2O,  deduct  the  Na2O  found  in  (1).     The  difference  is 
the  alkali  present  as  carbonate  expressed  in  terms  of  Na2O. 

4.  Alkali  combined  with   Organic  Acids.  —  The  sum  of  the 
percentages  of  Na2O  found  at  (1)  and  (3)  deducted  from  the 
percentage  found  at  (2)  is  a  measure  of  the  Na2O  or  other 
alkali  combined  with  organic  acids. 

5.  Determination  of  Acidity. — Take  10  grams  of  the  sample, 
dilute  with  50  cc.  distilled  water  free  from  carbon  dioxide,  ti- 
trate with  normal  sodium  hydroxide,  using  phenolphthalein  as 
indicator,  and  express  the  result  in  terms  of  Na2O  required  to 
neutralize  100  grams. 

6.  Determination  of  Total  Residue  at  160°  C. — For  this  de- 
termination the  crude  glycerin  should  be  slightly  alkaline  with 
sodium  carbonate,  not  exceeding  0.2  per  cent  Na2O  in  order  to 
prevent  loss  of  organic  acids.     To  avoid  the  formation  of  poly- 
glycerols,'  this  alkalinity  must  not  be  exceeded. 

Ten  grams  of  the  sample  are  put  in  a  100-cc.  flask,  diluted 
with  water,  and  the  calculated  quantity  of  normal  hydrochloric 
acid  or  sodium  carbonate  added  to  give  the  required  degree  of 
alkalinity.  The  flask  is  filled  to  100  cc.,  the  contents  mixed, 
and  10  cc.  measured  into  a  weighed  flat-bottomed  glass  dish  2.5 
inches  in  diameter  and  0.5  inches  deep.  In  the  case  of  crude 
glycerins  abnormally  high  in  organic  residue  a  smaller  amount 
should  be  taken  so  that  the  organic  residue  shall  not  materially 
exceed  30-40  milligrams. 

The  dish  is  placed  on  a  water  bath  (or  on  top  of  the  oven  kept 
at  160°)  until  most  of  the  water  has  evaporated,  then  placed  in 
an  oven  at  160°,  leaving  the  door,  of  the  oven  open  so  as  to  have 


SOAP   AND   GLYCERIN  279 

a  temperature  of  130°-140°,  until  the  glycerin,  or  most  of  it, 
has  evaporated.  When  only  a  slight  vapor  is  seen  to  come  off, 
the  dish  is  removed,  allowed  to  cool,  and  0.5  to  1  cc.  of  water 
added  and  by  a  rotary  motion  the  residue  brought  wholly  or 
nearly  into  solution.  The  dish  is  then  allowed  to  stand  on  top 
of  the  oven  until  the  water  has  evaporated  and  the  residue  is 
sufficiently  dry  to  prevent  spurting,  when  it  is  placed  in  the 
oven  at  160°  C.  The  dish  is  then  kept  in  the  oven  carefully 
maintained  at  160°  C.  for  one  hour,  when  it  is  removed,  cooled, 
the  residue  treated  with  water,  the  water  evaporated,  and  the 
residue  subjected  to  a  second  baking  of  one  hour,  after  which 
the  dish  is  allowed  to  cool  in  a  desiccator  over  sulphuric  acid 
and  weighed. 

The  treatment  with  water,  etc.,  is  repeated  until  a  constant 
loss  of  1  to  1.5  milligram  per  hour  is  obtained. 

In  the  case  of  acid  glycerin,  a  correction  must  be  made  for 
the  alkali  added,  1  cc.  normal  alkali  representing  an  addition  of 
0.03  gram  to  the  residue.  In  the  case  of  alkaline  glycerins  a 
correction  should  be  made  for  the  acid  added,  by  deducting  the 
increase  in  weight  due  to  the  conversion  of  sodium  hydroxide 
and  sodium  carbonate  to  sodium  chloride.  The  corrected  weight 
multiplied  by  100  gives  the  percentage  of  total  residue  at  160°  C. 

This  residue  is  used  for  determination  of  the  non-volatile 
acetylizable  impurities  as  described  under  the  acetin  method 
below. 

7.  Organic  Residues.  —  Subtract  the  ash  from  the  total  residue 
at  160°  C.  Report  as  organic  residue  at  160°  C.  (it  should  be 
noted  that  alkaline  salts  of  fatty  acids  are  converted  into  car- 
bonates on  ignition  and  that  the  carbon  dioxide  thus  derived  is 
not  included  in  the  organic  residue). 

G-lycerol  by  Acetin  Method 

This  process  is  the  one  agreed  upon  at  a  conference  of  dele- 
gates from  the  American,  British,  French,  and  German  Commit- 
tees, and  has  been  confirmed  by  each  of  the  above  committees 
as  giving  results  nearer  to  the  truth  than  the  dichromate 
method  on  crude  glycerins  in  general.  It  is  the  process  to  be 


280  METHODS    OF   ORGANIC   ANALYSIS 

used  (if  applicable)  whenever  only  one  method  is  employed. 
On  pure  glycerins  the  results  are  identical  with  those  obtained 
by  the  dichromate  process.  For  the  application  of  this  method 
the  crude  glycerin  should  not  contain  over  60  per  cent  water. 

Reagents.  —  (yl)  Best  acetic  anhydride.  —  This  should  be 
carefully  selected.  A  good  sample  must  not  require  more  than 
0.1  cc.  normal  sodium  hydroxide  for  saponification  of  the  im- 
purities in  a  blank  test  on  7.5  cc.  Only  a  slight  color  should 
develop  during  digestion  of  the  blank. 

The  anhydride  may  be  tested  for  strength  by  the  following 
method:  Into  a  weighed  stoppered  vessel,  containing  10  to  20 
cc.  of  water,  run  about  2  cc.  of  the  anhydride,  replace  the  stop- 
per, and  weigh.  Let  stand,  with  occasional  shaking,  for  several 
hours,  to  permit  the  hydrolysis  of  all  the  anhydride;  then  dilute 
to  about  200  cc.,  add  phenolphthalein,  and  titrate  with  normal 
sodium  hydroxide.  This  gives  the  total  acidity  due  to  free 
acetic  acid  and  acid  formed  from  the  anhydride.  It  is  worthy 
of  note  that  in  the  presence  of  much  free  anhydride  a  compound 
is  formed  with  phenolphthalein,  soluble  in  alkali  and  acetic 
acid,  but  insoluble  in  neutral  solutions.  If  a  turbidity  is 
noticed  toward  the  end  of  the  neutralization,  it  is  an  indication 
that  the  anhydride  is  incompletely  hydrolized,  and  inasmuch 
as  the  indicator  is  withdrawn  from  the  solution,  results  may  be 
incorrect. 

Into  a  stoppered  weighing  bottle  containing  a  known  weight  of 
recently  distilled  aniline  (from  10  to  20  cc.)  measure  about  2  cc. 
of  the  sample,  stopper,  mix,  cool,  and  weigh.  Wash  the  contents 
into  about  200  cc.  of  cold  water  and  titrate  the  acidity  as  before. 
This  yields  the  acidity  due  to  the  original  preformed  acetic  acid 
plus  one  half  the  acid  due  to  anhydride  (the  other  half  having 
formed  acetanilide) ;  subtract  the  second  result  from  the  first 
(both  calculated  to  100  grams)  and  double  the  result,  obtaining 
the  cubic  centimeters  of  normal  sodium  hydroxide  per  100  grams 
of  the  sample.  Each  cubic  centimeter  equals  0.0510  gram  an- 
hydride. 

(5)  Pure  fused  sodium  acetate. —  The  purchased  salt  is 
again  completely  fused  in  a  platinum,  silica,  or  nickel  dish, 


SOAP   AND   GLYCERIN  281 

avoiding  charring,  powdered  quickly  and  kept  in  a  stoppered 
bottle  or  desiccator.  It  is  important  that  the  sodium  acetate  be 
anhydrous. 

((7)  A  solution  of  sodium  hydroxide  for  neutralizing,  of 
about  normal  strength,  free  from  carbonate.  —  This  can  be 
readily  made  by  dissolving  pure  sodium  hydroxide  in  its  own 
weight  of  water  (preferably  free  from  carbon  dioxide)  and 
allowing  to  settle  until  clear  or  filtering  through  an  asbestos  or 
paper  filter.  The  clear  solution  is  diluted  with  water  free  from 
carbon  dioxide  for  the  strength  required. 

(D)  Normal  sodium  hydroxide  free  from  carbonate.  —  Pre- 
pared as  above  and  carefully  standardized.  Some  sodium  hy- 
droxide solutions  show  a  marked  diminution  in  strength  after 
being  boiled;  such  solutions  should  be  rejected. 

(.Z7)    Normal  acid  carefully  standardized. 

(^)  Phenolphthalein  solution,  a  one  half  per  cent  solution  in 
alcohol,  neutralized. 

Method. — In  a  narrow-mouthed  flask  (preferably  round 
bottom),  capacity  about  120  cc.,  which  has  been  thoroughly 
cleaned  and  dried,  weigh  accurately  and  as  rapidly  as  possible 
1.25  to  1.5  grams  of  the  glycerin.  A  Grethan  or  Lunge  pipette 
will  be  found  convenient.  Add  about  3  grams  of  the  anhy- 
drous sodium  acetate,  then  7.5  cc.  of  the  acetic  anhydride,  and 
connect  the  flask  with  an  upright  Liebig  condenser.  For  con- 
venience the  inner  tube  of  this  condenser  should  not  be  over  50 
cm.  long  and  9  to  10  mm.  inside  diameter.  The  flask  is  con- 
nected to  the  condenser  by  either  a  ground  glass  joint  (prefer- 
ably) or  a  rubber  stopper.  If  a  rubber  stopper  is  used,  it 
should  have  a  preliminary  treatment  with  hot  acetic  anhydride 
vapor.  Heat  the  contents  and  keep  just  boiling  for  one  hour, 
taking  precautions  to  prevent  the  salts  drying  on  the  sides  of 
the  flask.  Allow  the  flask  to  cool  somewhat,  and  through  the 
condenser  tube  add  50  cc.  of  distilled  water,  free  from  carbon 
dioxide,  at  a  temperature  of  about  80°  C.,  taking  care  that  the 
flask  is  not  loosened  from  the  condenser.  The  object  of  cool- 
ing is  to  avoid  any  sudden  rush  of  vapors  from  the  flask  on 
adding  water  and  to  avoid  breaking  the  flask.  Time  is  saved 


282  METHODS    OF   ORGANIC    ANALYSIS 

by  adding  the  water  before  the  contents  of  the  flask  solidifies, 
but  the  contents  may  be  allowed  to  solidify  and  the  test  pro- 
ceeded with  the  next  day  without  detriment,  bearing  in  mind 
that  the  anhydride  in  excess  is  much  more  effectively  hydro- 
lized  in  hot  than  in  cold  water.  The  contents  of  the  flask  may 
be  warmed  to,  but  must  not  exceed,  80°  C.,  until  the  solution  is 
complete  except  a  few  dark  flocks  representing  organic  impur- 
ities in  the  crude  glycerin.  By  giving  the  flask  a  rotary 
motion,  solution  is  more  quickly  effected. 

Cool  the  flask  and  contents  without  loosening  from  the  con- 
denser. When  quite  cold  wash  down  the  inside  of  the  con- 
denser tube,  detach  the  flask,  wash  off  the  stopper  or  ground 
glass  connection  into  the  flask,  and  filter  the  contents  through 
an  acid-washed  filter  into  a  Jena  glass  flask  of  about  one  liter 
capacity.  Wash  thoroughly  with  cold  distilled  water,  free 
from  carbon  dioxide.  Add  2  cc.  of  phenolphthalein  solution 
(-F),  then  run  in  caustic  soda  solution  (  (7)  or  (D)  until  a  faint 
pinkish  yellow  color  appears  throughout  the  solution.  This 
neutralization  must  be  done  most-  carefully  ;  the  alkali  should 
be  run  down  the  sides  of  the  flask,  the  contents  of  which  are 
kept  rapidly  swirling  with  occasional  agitation  or  change  of 
motion  until  the  solution  is  nearly  neutralized,  as  indicated  by 
the  slower  disappearance  of  the  color  developed  locally  by  the 
alkali  running  into  the  mixture.  When  this  point  is  reached, 
the  sides  of  the  flask  are  washed  down  with  carbon-dioxide-free 
water,  and  the  alkali  subsequently  added  drop  by  drop,  mixing 
after  each  drop  until  the  desired  tint  is  obtained. 

Now  run  in  from  a  burette  50  cc.  or  a  calculated  excess  of 
normal  sodium  hydroxide  {D)  and  note  carefully  the  exact 
amount.  Boil  gently  for  fifteen  minutes,  the  flask  being  fitted 
with  a  glass  tube  acting  as  a  partial  condenser.  Cool  as 
quickly  as  possible  and  titrate  the  excess  of  sodium  hydroxide 
with  normal  acid  (J7)  until  the  pinkish  yellow  er  chosen  end 
point  color  just  remains.1 

1  A  precipitate  at  this  point  is  an  indication  of  the  presence  of  iron  or  alu- 
minium and  high  results  will  be  obtained  unless  a  correction  is  made  as  described 
below. 


SOAP   AND    GLYCERIN  283 

A  further  addition  of  the  indicator  at  this  point  will  cause 
an  increase  of  the  pink  color;  this  must  be  neglected  and  the 
first  end  point  taken. 

From  the  amount  of  normal  sodium  hydroxide  consumed, 
calculate  the  percentage  of  glycerol  (including  acetylizable 
impurities)  after  making  correction  for  the  blank  test  described 
below. 

1  cc.  normal  sodium  hydroxide  corresponds  to  0.03069  gram 
glycerol. 

•  The  coefficient  of  expansion  for  normal  solutions  is  0.00033 
per  cubic  centimeter  for  each  degree  C.  A  correction  should 
be  made  on  this  account,  if  necessary. 

Blank  Test.  —  As  the  acetic  anhydride  and  sodium  acetate 
may  contain  impurities  which  affect  the  result,  it  is  necessary 
to  make  a  blank  test,  using  the  same  quantities  of  acetic 
anhydride,  sodium  acetate,  and  water  as  in  the  analysis.  It  is 
not  necessary  to  filter  the  solution  of  the  melt  in  this  case,  but 
sufficient  time  must  be  allowed  for  the  hydrolysis  of  the 
anhydride  before  proceeding  with  the  neutralization.  After 
neutralization  it  is  not  necessary  to  add  more  than  10  cc.  of 
normal  alkali  (-0),  as  this  represents  the  excess  usually  present 
after  the  saponification  of  the  average  soap  lye  crude  glycerin. 
In  determining  the  acid  equivalent  of  the  normal  sodium 
hydroxide,  however,  the  entire  amount  taken  in  the  analysis, 
50  cc.,  should  be  titrated  after  dilution  with  300  cc.  of  water 
free  from  carbon  dioxide  and  without  boiling. 

Determination  of  the  Crlycerol  Value  of  the  Acetylizable  Im- 
purities. —  The  total  residue  at  160°  C.  is  dissolved  in  one  or 
two  cubic  centimeters  of  water,  washed  into  the  acetylizing 
flask,  and  evaporated  to  dryness.  Then  add  anhydrous  sodium 
acetate  and  acetic  anhydride  in  the  usual  amounts  and  proceed 
as  described  in  the  regular  analysis.  After  correcting  for  the 
blank  calculate  the  result  to  glycerol. 

Calculation  of  the  Actual  Crlycerol  Contents.  —  (1)  Determine 
the  apparent  percentage  of  glycerol  in  the  sample  by  the  acetin 
process  as  described.  The  result  will  include  acetylizable 
impurities,  if  any  are  present. 


284  METHODS    OF   ORGANIC    ANALYSIS 

(2)  Determine  the  total  residue  at  160°  C. 

(3)  Determine  the  acetin  value  of  this  residue  in  terms  of 
glycerol. 

(4)  Deduct  the  result  found  at   (3)  from  the  percentage 
obtained  at  (1)  and  report  this  corrected  figure  as  glycerol.     If 
volatile  acetylizable  impurities  are  present,  these  are  included 
in  this  figure. 

Trimethyleneglycol  is  more  volatile  than  glycerol  and  can 
therefore  be  concentrated  by  fractional  distillation.  An  ap- 
proximation to  the  quantity  can  be  obtained  from  the  differ- 
ence between  the  results  by  the  acetin  and  by  the  dichromate 
method  on  such  distillates.  The  difference  multiplied  by  1.736 
will  give  the  glycol. 

Crlycerol  by  Dichromate  Method 

Reagents.  —  {A)  Pure  potassium  dichromate  powdered  and 
dried  in  a'ir  free  from  dust  or  organic  vapors  at  110°  to  120°  C. 
This  is  taken  as  the  standard. 

(.#)  Dilute  dichromate  solution.  —  7.4564  grams  of  the 
above  dichromate  are  dissolved  in  distilled  water  and  the  solu- 
tion made  up  to  one  liter  at  15.5°  C. 

((7)  Ferrous  ammonium  sulphate. — It  is  never  safe  to  as- 
sume this  salt  to  be  constant  in  composition  and  it  must  be 
standardized  against  the  dichromate  as  follows :  Dissolve 
3.7282  grams  of  dichromate  (A)  in  50  cc.  of  water.  Add 
50  cc.  of  50  per  cent  sulphuric  acid  (by  volume)  and  to  the 
cold  undiluted  solution  add  from  a  weighing  bottle  a  moderate 
excess  of  the  ferrous  ammonium  sulphate  and  titrate  back  with 
the  dilute  dichromate  (-5).  Calculate  the  value  of  the  ferrous 
salt  in  terms  of  dichromate. 

(-Z>)  Silver  carbonate. — This  is  prepared  as  required  for 
each  test  from  140  cc.  of  0.5  per  cent  silver  sulphate  solution 
by  precipitation  with  about  4.9  cc.  normal  sodium  carbonate 
solution  (a  little  less  than  the  calculated  quantity  of  sodium 
carbonate  should  be  used,  as  an  excess  prevents  rapid  settling). 
Settle,  decant,  and  wash  once  by  decantation. 

(JS)  Subacetate  of  lead.  —  Boil  a  10  per  cent  solution  of  pure 


.    SOAP   AND    GLYCERIN  285 

lead  acetate  with  an  excess  of  litharge  for  one  hour,  keeping  the 
volume  constant,  and  filter  while  hot.  Disregard  any  precipi- 
tate which  subsequently  forms.  Preserve  out  of  contact  with 
carbon  dioxide. 

(jP)  Potassium  ferricyanide.  —  A  very  dilute  freshly  pre- 
pared solution  containing  about  0.1  per  cent. 

Method.  —  Weigh  20  grams  of  the  glycerin,  dilute  to  250  cc., 
and  take  25  cc.  Add  the  silver  carbonate,  allow  to  stand  with 
occasional  agitation  for  about  ten  minutes,  and  add  a  slight 
excess  (about  5  cc.  in  most  cases)  of  the  basic  lead  acetate  (^E) ; 
allow  to  stand  a  few  minutes,  dilute  with  distilled  water  to 
100  cc.  and  then  add  0.15  cc.  to  compensate  for  the  volume  of 
the  precipitate ;  mix  thoroughly,  filter  through  an  air-dry  filter 
into  a  suitable  narrow-mouthed  vessel,  rejecting  the  first  10  cc., 
and  return  the  filtrate,  if  not  clear  and  bright.  Test  a  portion 
of  the  filtrate  with  a  little  basic  lead  acetate,  which  should 
produce  no  further  percipitate  (in  the  great  majority  of  cases 
5  cc.  are  ample,  but  occasionally  a  crude  glycerin  will  be  found 
requiring  more,  and  in  this  case  another  portion  of  25  cc.  of 
the  dilute  glycerin  should  be  taken  and  purified  with  6  cc.  of 
the  basic  acetate).  Care  must  be  taken  to  avoid  a  marked 
excess  of  basic  acetate. 

Measure  off  25  cc.  of  the  clear  filtrate  into  a  flask  or  beaker, 
previously  cleaned  with  potassium  dichromate  arid  sulphuric 
acid.  Add  12  drops  of  sulphuric  acid  (1  to  4)  to  precipitate 
the  small  excess  of  lead  as  sulphate.  Add  3.7282  grams  of 
the  powdered  potassium  dichromate  (^1).  Rinse  down  the 
dichromate  with  25  cc.  of  water  and  let  stand  with  occasional 
shaking  until  all  the  dichromate  is  dissolved  (no  reduction  will 
take  place  in  the  cold). 

Now  add  50  cc.  of  50  per  cent  sulphuric  acid  (by  volume) 
and  immerse  the  vessel  in  boiling  water  for  two  hours  and 
keep  protected  from  dust  and  organic  vapors,  such  as  alcohol, 
until  the  titration  is  completed.  Add  from  a  weighing  bottle 
a  slight  excess  of  the  ferrous  ammonium  sulphate  ((7),  making 
spot  tests  on  a  porcelain  plate  with  the  potassium  ferricyanide 
(-F).  Titrate  back  with  the  dilute  dichromate.  From  the 


286  METHODS  OF  ORGANIC  ANALYSIS 

amount   of   dichromate   reduced,    calculate   the  percentage  of 
glycerol. 

One  gram  glycerol        =  7.4564  grains  dichromate 
One  gram  dichromate  =  0.13411  gram  glycerol. 

The  percentage  of  glycerol  obtained  above  includes  any 
oxidizable  impurities  present  after  purification.  A  correction 
for  the  non-volatile  impurities  may  be  made  by  running  a  di- 
chromate test  on  the  residue  at  160°  C. 

Notes.  —  (1)  It  is  important  that  the  concentration  of  acid 
in  the  oxidation  mixture  and  the  time  of  oxidation  should  be 
strictly  adhered  to. 

(2)  Before  the  dichromate  is  added  to  the  glycerin  solution 
it  is  essential  that  the  slight  excess  of  lead  be  precipitated  with 
sulphuric  acid  as  stipulated. 

(3)  For  crude  glycerins  practically  free  from  chlorides  the 
quantity  of  silver  carbonate  may  be  reduced  to  one-fifth  and 
the  basic  lead  acetate  to  0.5  cc. 

(4)  It  is  sometimes  advisable  to  add  a  little  potassium  sul- 
phate to  insure  a  clear  filtrate. 

An  appendix  to  the  committee  report  from  which  the  above 
methods  are  taken  describes  and  illustrates  a  new  form  of 
sampling  tube  for  taking  samples  of  crude  glycerin  from 
drums. 

REFERENCES 

I 
ALDER-WRIGHT  and  MITCHELL  :  Animal  and  Vegetable  Fixed  Oils,  Fats, 

Butters,  and  Waxes. 

ALLEN  :  Commercial  Organic  Analysis,  Vol.  II. 
BENEDIKT-ULZER  :  Analyse  der  Fette  und  Wachsarten. 
LAMBORN:  Modern  Soaps,  Candles,  and  Glycerin. 

LEWKOWITSCH  :  Chemical  Technology  and  Analysis  of  the  Oils,  Fats,  and 
Waxes. 

II 

1900.    DEVINE:  A  Method  of  Determining  Free  Alkali  in  Soaps.     J.  Am. 

Chem.  Soc.,  22,  693. 

HENRIQUES  and  MAYER  :    (Determination  of  Total,  Free,  and  Car- 
bonated Alkali  in  Soaps).     Z.  angew.  Chem.,  1900,  785. 

1902.   DOANE  :  The  Disinfectant  Properties  of  Washing  Powders.     Bui.  79, 
Maryland  Agricultural  Experiment  Station. 


SOAP   AND   GLYCERIN  287 

FRIEDRICH:  (Soap  Analysis)  4  Bericht  des  Vereins  gegen  Verfal- 
schung  der  Lebensmittel,  etc.,  Chemnitz,  1902,  132 ;  Z.  Ndhr. 
Genussm.,  1903,  6,  851. 

1903.  HELLER:  (Significance  of  Soaps  in  Disinfectants).     Arch.  Hygiene, 

47,  213. 

1904.  HEERMANN:    Determination  of   Caustic  and  Carbonated  Alkali  in 

Soaps.     Chem.  Ztg.,  28,  531. 
MARTIN  :  Determination  of  Glycerol  in  Soap.     Moniteur  Scientifique, 

[4],  17,  797. 
VAN  SLYKE  and  URNER  :  The  Composition  of  Commercial  Soaps  in 

Relation  to  Spraying.     Bui.  257,  New  York  State  Agricultural 

Experiment  Station. 

1905.  DEVINE  :  Determination  of  Rosin  in  Soaps.     Chem.  Eng.,  1,  207. 
MATTHEWS  :  The  Effects  of  Alkaline  Scouring  Agents  on  the  Strength 

of  Woolen  Yarns.     /.  Soc.  Chem.  Ind.,  24,  659. 

RODET:  Experiments  on  the  Antiseptic  Value  of  Common  Soaps. 
Revue  d' Hygiene,  27,  301. 

1907.  JACKSON  :  Detergents  and  Bleaching  Agents  used  in  Laundry  Work. 

J.  Soc.  Arts,  55,  1101,  1122 ;  Chem.  Abs.,  2,  327. 

1908.  BORNEMANN  :  Fat,  Soap,  and  Candle  Industry  in  1907.     Chem.  Ztg., 

32,  741,  755. 
FENDLER  and  FRANK:    Determination  of  Fatty  Acid  Content   of 

Soaps.     Z.  angew.  Chem.,  22,  252. 
STEINER  :  (Recent  Development  of  the  Soap  Industry).     Chem.  Ztg.j 

32,  445,  458. 

1909.  DOMINIKIEWICZ  :  New  Method  for  Determination  of  Fatty  Acids  in 

Soap.     Chem.  Ztg.,  33,  728. 

SY  :  Mercury  Seals  in  Fat  Extraction  Apparatus  and  a  New  Form  of 
Flask.  J.  Ind.  Eng.  Chem.,  1,  314. 

1910.  COMEY  and  BACKUS  :  The  Coefficient  of  Expansion  of  Glycerin.     /. 

Ind.  Eng.  Chem.,  2, 11. 

1911.  Committee  Report  on  Glycerin  Analysis.     J.  Ind.  Eng.  Chem.,  3,  679. 
HAMILTON  :  Soaps  from  Different  Glycerides ;  their  Germicidal  and 

Insecticidal  Values  Alone  and  Associated  with  Active  Agents. 
J.  Ind.  Eng.  Chem.,  3,  582. 


CHAPTER   XIV 

Nitrogen,  Sulphur,  and  Phosphorus 
THE  DETERMINATION  OF  NITROGEN 

THE  well-known  copper  oxide  method  of  Duinas,  sometimes 
called  the  absolute  method,  has/the  advantage  of  being  applicable 
to  all  classes  of  nitrogen  compounds.  In  the  great  majority  of 
cases,  however,  it  is  equally  accurate  and  much  more  conven- 
ient to  use  one  of  the  methods  based  upon  the  conversion  of 
nitrogen  to  ammonia  and  the  determination  of  the  latter  by 
titration. 

Before  the  introduction  of  the  Kjeldahl  process,  the  soda- 
lime  method  of  Will  and  Varrentrap  was  commonly  used.  In 
this  method  the  finely  ground  substance  is  mixed  with  a  large 
excess  of  soda  lime  and  heated  in  a  combustion  tube,  the  am- 
monia given  off  being  absorbed  in  standard  acid.  In  order  to 
insure  the  reduction  of  nitro-compounds  or  nitrates,  this  method 
was  modified  by  the  introduction  of  stannous  sulphide  (Gold- 
berg) ;  of  sodium  formate  and  sodium  thiosulphate  (Arnold)  ; 
or  of  sodium  thiosulphate  and  a  mixture  of  equal  parts  sulphur 
and  powdered  sugar  or  charcoal  (Ruffle).  The  soda-lime 
method  has  now  been  very  generally  superseded  by  the  various 
modifications  of  the  Kjeldahl  process. 

THE  KJELDAHL  METHOD 

In  this  process  the  substance  is  decomposed  by  heating  with 
strong  sulphuric  acid,  usually  with  the  addition  of  some  reagent 
which  assists  the  decomposition  either  by  raising  the  boiling 
point  or  by  acting  as  a  carrier  of  oxygen.  When  decomposition 
is  complete,  the  nitrogen  remains  as  ammonium  sulphate  in  the 
sulphuric  acid,  the  carbon  and  hydrogen  of  the  substance  hav- 

288 


NITROGEN,    SULPHUR,    AND    PHOSPHORUS  289 

ing  been  oxidized  and  the  products  of  oxidation  boiled  out  of 
the  solution.  The  oxidation  takes  place  partly  at  the  expense 
of  the  sulphuric  acid,  so  that  a  considerable  evolution  of  sulphur 
dioxide  occurs,  especially  in  the  earlier  stages  of  the  process. 
After  the  completion  of  the  digestion,  the  ammonia  is  liberated 
by  means  of  fixed  alkali  and  determined  by  distilling  into 
standard  acid.  In  the  va^gus  modifications  of  the  process, 
different  reagents  or  combinations  of  reagents  are  used  to  hasten 
the  decomposition  of  the  organic  matter  by  the  sulphuric  acid. 

Kjeldahl  originally  directed  *  that  the  substance  be  heated 
with  sulphuric  acid,  with  or  without  the  addition  of  phosphoric 
anhydride,  until  a  clear  solution  is  obtained ;  then  potassium 
permanganate  added  in  small  portions  to  the  hot  solution  until 
it  remains  permanently  colored,  the  permanganate  being  added 
very  cautiously  on  account  of  the  danger  of  a  loss  of  nitrogen 
if  the  reaction  becomes  too  vigorous. 

Willfarth  2  introduced  the  use  of  mercuric  oxide  to  facilitate 
the  action  of  sulphuric  acid,  and  stated  that  if  the  solution  be 
boiled  until  colorless,  neither  phosphoric  anhydride  nor  potas- 
sium permanganate  need  be  used.  In  his  earlier  experiments 
Willfarth  used  copper  instead  of  mercury.  Arnold  3  used  both 
mercury  and  copper  in  addition  to  phosphoric  anhydride. 

Gunning  *  used  for  the  digestion  a  simple  mixture  of  sulphuric 
acid  with  one  third  to  one  half  its  weight  of  potassium  sulphate. 
Arnold  and  Wedemeyer  5  modified  the  Gunning  process  by  the 
use  of  mercury  and  copper  in  addition  to  the  potassium  sulphate. 
By  this  modification  the  time  required  to  decompose  the  organic 
matter  was  greatly  reduced  and  good  results  were  obtained 
with  a  number  of  alkaloids  and  other  compounds  which  had 
not  readily  yielded  the  whole  of  their  nitrogen  when  treated 
by  the  methods  previously  used.  Independently  Dyer6  ob- 
tained equally  good  results  on  a  wide  range  of  organic  com- 

1  Z.  anal.  Chem.,  1883,  22,  366. 

2  Chem.  ZentrbL,  1885,  [3],  16,  17,  113. 

3  Archw.  der  Pharm.,  [3],  24,  785  ;  Z.  anal.  Chem.,  26,  249. 

*Z.  anal,  Chem.,  1889,  28,  188.  5  Z.  anal.  Chem.,  1892,  31,  525. 

6  J.  Chem.  Soc.,  1895,  67,  811. 
u 


290  METHODS    OF   ORGANIC    ANALYSIS 

pounds  by  the  use  of  mercury  and  potassium  sulphate  without 
copper. 

The  Official  Agricultural  Chemists  authorize  l  the  Kjeldahl- 
Willfarth  and  the  Gunning  methods  for  the  analysis  of  foods, 
spices  other  than  peppers,  and  fertilizers  not  containing  nitrates. 
For  peppers,  to  secure  complete  ammonification  of  the  alkaloidal 
nitrogen,  the  Arnold-Wedemeyer  modification  of  the  Gunning 
method  was  provisionally  adopted  in  1902.  In  the  experience  of 
this  laboratory,  the  Dyer  modification  has  been  found  more  rapid 
and  slightly  more  accurate  than  either  the  Kjeldahl-Willfarth  or 
Gunning  method  and  fully  as  efficient  in  the  case  of  alkaloids 
as  is  the  Arnold-Wedemeyer  method,  while  it  has  the  advantage 
over  the  latter  of  requiring  one  less  reagent  and  of  yielding  a 
colorless  solution. 

Applicability  of  the  Kjeldahl  Method.  — The  Gunning- Arnold- 
Dyer  modification,  which  for  the  reasons  just  mentioned  is  rec- 
ommended for  general  use,  is  applicable  to  all  classes  of  animal 
and  vegetable  substances,  including  such  difficultly  decomposable 
bases  as  betaine  and  pyridine  and  chinoline  alkaloids,  and  to 
cyanides,  ferrocyanides,  and  ferricyanides.  It  has  also  been 
tested2  with  good  results  on  many  other  compounds,  including 
acetanilid,  sulphanilic  acid,  orthobenzoic  sulphinid,  aminoben- 
zoic  acid,  benzamid,  diaminophenol,  naphthylamine,  diphenyl- 
amine,  diphenylthiourea,  nitroso-dimethylaniline,  indigotin, 
pyridine,  and  oxyphenyl  methylpyrimidine. 

Jodlbauer's  modification,3  devised  for  the  determination  of 
nitrogen  in  nitrates,  was  found  by  Dyer  to  be  applicable  to  nitro- 
compounds,  to  azo-,  hydrazo-,  and  amidoazo-benzene,  to  carbazol, 
and,  with  the  addition  of  1  or  2  grams  of  sugar,  to  hydroxyl- 
amine  and  oximes.  Dyer  did  not  obtain  the  whole  of  the  nitro- 
gen of  hydrazine  derivatives,  but  Dafert 4  and  Milbauer  6  have 
published  modifications  which  are  said  to  give  accurate  results 
with  this  class  of  compounds. 

1  Bulletin  107,  Revised,  Bureau  of  Chemistry,  U.  S.  Dept.  Agriculture. 

2  In  some  cases  without  the  use  of  mercury.     See  in  addition  to  the  papers  al- 
ready cited,  that  of  Gibson  in  J.  Am.  Chem.  Soc.,  1904,  26,  105. 

8  Chem.  Zentrbl.,  1886,  3,  17,  433. 

*Landw.  Versuchs-Sta.,  1887,  34,  311.          5  Z.  anal.  Chem.,  1903,  42,  725. 


NITROGEN,    SULPHUR,    AND    PHOSPHORUS 


291 


GUNNING-ARNOLD-DYER    MODIFICATION 

Reagents.  —  Pure  concentrated  sulphuric  acid.  Mercury. 
Pure  potassium  sulphate.  Potassium  sulphide  solution,  40  grams 
per  liter.  Saturated  solution  of  sodium  hydroxide  (commercial) . 
Granulated  zinc  or  pumice  stone.  Paraffin.  Solution  of  methyl 
orange,  congo  red,  cochineal,  lacmoid,  or  any  other  indicator 
suitable  for  titration  in  the  presence  of  ammonium  salts. 
Standard  solutions  of  acid  and  al- 
kali, preferably  one  fifth  or  one 
tenth  normal. 

Determination.  —  Weigh  0.5  to 
5.0  grams  sample,  or  so  much  as 
will  probably  yield  from  50  to  75 
milligrams  of  ammonia,  and  trans- 
fer to  a  pear-shaped  Kjeldahl  flask 
of  550  to  750  cc.  capacity.  Add  20 
to  25  cc.  concentrated  sulphuric 
acid  and  about  0.7  gram  of  mer- 
cury. Place  the  flask  in  an  in- 
clined position  (Fig.  16)  and  heat 
gently  until  the  first  vigorous 
frothing  ceases,  then  raise  the  heat 
gradually  until  the  liquid  boils  ; 
remove  the  flame  for  a  few  minutes, 
add  10  to  12  grams  of  potassium 
sulphate,  and  boil.  If  the  liquid 

-,  n        T     .,.  .,         .,,          FIG.  16.  — Kjeldahl  digestion  flask 

IS    kept    actually    boiling,    it    Will     in  position  on  ordinary  ring  stand. 

usually    be    clear    and    colorless 

within  30  minutes  after  the  addition  of  the  sulphate.  Con- 
tinue the  boiling  for  at  least  30  minutes  after  the  solution 
becomes  colorless  or  in  any  case  for  one  hour  from  the  time 
the  potassium  sulphate  is  added.  If  the  sample  contains  al- 
kaloids, the  boiling  should  be  continued  for  at  least  three  hours 
in  all  and  not  less  than  two  hours  after  the  solution  is  colorless. 
When  the  digestion  is  finished,  allow  the  flask  to  cool  for  10  to 
15  minutes  or  to  40°-60°  (if  allowed  to  become  thoroughly  cold 


*_* 


292 


METHODS  OF  ORGANIC  ANALYSIS 


the  solution  solidifies)  ;  then  dilute  carefully  with  150  to  200 
cc.  of  water  ;  allow  to  cool,  add  25  cc.  potassium  sulphide  solu- 
tion, mix  well,  and  then  add  75  to  100  cc.  (or  enough  to  make 
the  reaction  strongly  alkaline  -1)  of  a  cold  saturated  solution  of 
sodium  hydroxide,  pouring  it  carefully  down  the  side  of  the  flask, 
so  that  it  does  not  mix  immediately  with  the  acid  solution.  Add 
a  few  pieces  of  granulated  zinc  or  pumice  stone  to  prevent  bump- 
ing, and  a  piece  of  paraffin  the  size  of  a  pea  to  diminish  frothing  ; 
connect  the  flask,  preferably  by  means  of  a  Hopkins  distilling 
head  (Fig.  17),  with  a  condenser,  the  deliv- 
ery tube  of  which  dips  into  50  cc.  of  tenth 
normal  sulphuric  or  hydrochloric  acid  or  its 
equivalent  in  the  receiver  ;  mix  the  contents 
of  the  flask  by  shaking,  and  distill  until  about 
one  half  of  the  liquid  has  passed  into  the  re- 
ceiver. Titrate  the  excess  of  acid  in  the 
receiver  by  means  of  standard  alkali,  using 
one  of  the  indicators  mentioned  above. 

The  reagents  used  should  be  tested  by 
making  a  blank  determination  with  pure 
sugar  or  cellulose,  carrying  out  all  opera- 
tions in  exactly  the  same  way  as  in  a  regular 
analysis. 

Notes.  —  If  in  transferring  the  substance  to  the  flask  any  par- 
ticles or  drops  should  lodge  in  the  neck,  they  can  be  washed 
down  by  the  sulphuric  acid  subsequently  added.  Dry  samples 
can  usually  be  weighed  on  a  watch  glass  and  brushed  into  the 
flask  through  a  funnel  having  a  wide  stem  which  has  been  cut 
down  to  a  length  of  about  1  cm.,  or  transferred  by  means  of 
a  narrow  strip  of  glazed  paper.  It  is  often  more  convenient  to 
weigh  the  sample  on,  or  transfer  to,  a  small  piece  of  pure  filter 
paper,  fold  the  latter  loosely  over  the  weighed  portion,  and  in- 
troduce the  whole  into  the  flask  in  such  a  way  that  the  sample 
can  be  easily  shaken  free  from  the  paper.  The  amount  of  cel- 
lulose thus  introduced  with  the  sample  does  not  materially  pro- 

1  Corallin  (rosolic  acid)  may  be  used  as  indicator  to  show  that  the  contents 
of  the  flask  are  alkaline  before  beginning  the  distillation.  - 


FIG.  17.— The  Hop- 
kins distilling  head  in 
position. 


NITROGEN,    SULPHUR,    AND    PHOSPHORUS  293 

long  the  time  required  for  digestion,  while  its  reducing  effect 
may  aid  in  the  ammonificatioii  of  any  firmly  bound  nitrogen 
present.  Time  should  be  allowed  for  thorough  wetting  of  the 
sample  by  the  sulphuric  acid  before  heat  is  applied.  During 
digestion  a  small  funnel  or  balloon  stopper  may  be  placed 
loosely  in  the  mouth  of  the  flask  to  retard  the  evaporation  of 
acid  and  guard  against  mechanical  loss.  The  flask  may  rest 
upon  a  wire  gauze,  an  iron  plate,  or  a  piece  of  asbestos  having  a 
circular  hole  about  4  cm.  in  diameter  which  permits  the  free 
flame  of  the  Bunsen  burner  to  play  upon  the  flask  below  the 
surface  of  the  boiling  liquid.  The  digestion  should  be  con- 
ducted in  a  well-ventilated  hood  where  the  air  is  free  from  any 
considerable  amount  of  ammonia.  In  order  to  hasten  the  de- 
composition of  organic  matter,  Dakin1  and  Milbauer  (1.  <?.) 
recommend  the  use  of  potassium  persulphate,  while  Kriiger2 
suggests  the  addition  of  potassium  dichromate  in  excess  of  the 
amount  necessary  to,,  oxidize  all  carbon  and  hydrogen  present. 
The  method  here  described  is,  However,  sufficiently  rapid  and 
more  convenient  for  ordinary  work. 

The  precipitation  of  mercury  as  sulphide  before  rendering  the 
solution  alkaline  is  to  prevent  the  formation  of  mercur-ammo- 
nium  compounds  which  do  not  readily  yield  their  ammonia  on 
boiling  with  caustic  alkali.  Ai\  ordinary  Liebig  condenser 
can  be  used  in  this  distillation,  though  block  tin  tubing  is  gener- 
ally preferred  as  being  more  resistant  to  the  action  of  steam  and 
ammonia.  Unless  the  condenser  tube  is  straight  and  nearly 
vertical,  it  is  safer  to  rinse  it  out  with  a  little  cold  distilled 
water  at  the  end  of  the  distillation  and  add  the  rinsings  to  the 
distillate.  The  distillation  as  usually  carried  out  requires  from 
forty  minutes  to  one  hour.  Only  about  one  third  of  this  time 
is  actually  required  to  expel  the  ammonia  from  the  boiling 
solution,  and  if  it  is  desired  to  hasten  the  operation,  the  steam 
can  be  conducted  through  tin  tubing  without  condensation  into 
a  cooled  receiver  ;  or,  as  suggested  by  Benedict 3  the  water 
which  cools  the  condenser  may  be  drawn  off  after  the  solution 

!</.  Soc.  Chem.  Ind.,  1902,  21,  848. 

*Ber.,  1894,  27,  609.  3  J.  Am.  Chem.  Soc.,  1900,  22,  259. 


294  METHODS    OF   ORGANIC    ANALYSIS 

has  been  boiling  15  minutes,  and  the  boiling  continued  until 
enough  steam  has  passed  through  to  carry  all  of  the  ammonia 
from  the  condenser  tube  into  the  receiver.  Fixed  alkali  is 
preferable  to  ammonia  for  the  final  titration,  since  the  latter 
reagent  is  liable  to  lose  strength  during  use,  probably  through 
evaporation  of  ammonia  from  the  tip  of  the  burette  and  from 
the  falling  drop. 

The  reasons  for  preferring  the  method  as  above  described  to 
the  original  Kjeldahl-Willfarth  or  the  Gunning  method  have 
been  briefly  outlined  above.  If  either  of  the  latter  methods  is 
used,  the  same  details  of  manipulation  may  be  followed  except 
that  the  digestion  should  be  continued  for  at  least  two  hours 
after  the  solution  becomes  colorless  and  not  less  than  three 
hours  in  all.  Even  with  this  longer  time  of  boiling  the  results 
are  often  slightly  low.  For  the  results  of  a  comparative  study 
of  the  Kjeldahl-Willfarth,  the  Gunning,  and  the  above  described 
method  with  special  reference  to  the  time  of  boiling  required 
see  the  Journal  of  the  America1^  Chemical  Society  for  April  and 
November,  1904. 

METHOD  FOR  NITRATES  AND  NITRO-COMPOUNDS 

The  loss  of  nitric  acid  which  might  otherwise  occur  when 
sulphuric  acid  is  poured  upon  a  sample  containing  nitrates  is 
avoided  by  having  present  some  substance  which  is  very  easily 
nitrated,  such  as  salicylic  acid,  benzoic  acid,  or  phenol.  The 
nitrogen  having  been  thus  fixed  as  a  nitro-compound,  the  lat- 
ter may  be  reduced  by  the  addition  of  zinc  dust  (Jodlbauer)  or 
sodium  thiosulphate  (Forster),  after  which  the  determination  is 
carried  out  in  the  usual  manner. 

Reagents.  — Sulphuric-salicylic  acid,  prepared  in  advance  by 
dissolving  salicylic  acid  in  pure  concentrated  sulphuric  in  the 
proportion  of  2  grams  of  the  former  to  30  cc.  of  the  latter. 
Zinc  dust,  an  impalpable  powder ;  or  crystallized  sodium  thio- 
sulphate. Other  reagents  as  for  the  method  above  described. 

Determination.  —  To  the  weighed  portion  of  substance  in  a 
Kjeldahl  flask,  add  30  cc.  of  the  sulphuric-salicylic  acid  solu- 
tion and  shake  until  thoroughly  mixed ;  then  add  5  grains 


NITROGEN,    SULPHUR,    AND   PHOSPHORUS  295 

crystallized  sodium  thiosulphate,  or  add  gradually  with  con- 
stant shaking  2  grams  of  zinc  dust ;  warm  gently  until  froth- 
ing subsides  and  then  boil  until  thick  white  fumes  cease  to  be 
given  off.  Allow  to  cool  somewhat,  add  mercury  and  potas- 
sium sulphate,  and  complete  the  process  as  described  above. 
Or  the  potassium  sulphate  may  be  omitted  and  the  oxidation 
completed  by  means  of  permanganate  as  in  the  "  modified  Kjel- 
dahl "  method  of  the  Official  Agricultural  Chemists. 

Notes.  —  Thiosulphate  is  the  more  convenient  reducing  agent 
and  can  be  safely  used  if  the  amount  of  nitric  nitrogen  is  small. 
Zinc  dust  appears  to  be  a  safer  reagent  for  samples  rich  in  ni- 
trates. In  the  presence  of  considerable  amounts  of  chloride  or 
of  ammonium  salts  there  is  danger  of  a  loss  of  nitrogen  as  ox- 
ide when  the  sample  is  treated  with  the  sulphuric-salicylic  acid 
mixture.  In  such  cases  the  latter  mixture  should  be  thoroughly 
cooled  before  using  and  then  poured  as  quickly  as  possible  over 
the  sample  and  the  flask  allowed  to  stand  with  occasional  shak- 
ing for  at  least  half  an  hour  before  the  reducing  agent  is  added. 
Ten  minutes  should  be  allowed  for  the  reduction  to  take  place 
before  the  solution  is  heated.  With  these  precautions  the  loss 
of  nitrogen  will  be  reduced  to  an  amount  too  small  to  be  appre- 
ciable in  ordinary  work. 

THE  DETERMINATION  OF   SULPHUR 

In  the  various  methods  employed  for  the  determination  of  sul- 
phur, organic  matter  is  destroyed  by  oxidation  and  the  result- 
ing sulphuric  acid  is  precipitated  and  weighed  as  barium  sulphate 
in  the  usual  way.  The  chief  difficulty  lies  in  securing  complete 
oxidation  without  loss,  since  many  substances  contain  sulphur  in 
forms  from  which  it  is  easily  liberated  as  volatile  compound. 

COMPARATIVE  OUTLINE  OF  METHODS 

The  principal  methods  of  bringing  about  the  oxidation  of 
the  organic  matter  are  : 

1.  By  heating  with  nitric  acid  or  an  oxidizing  acid  mixture. 

2.  By  burning  in  a  combustion  tube  in  a  current  of  air,  oxy- 
gen, or  some  oxidizing  vapor. 


296  METHODS   OF   ORGANIC   ANALYSIS 

3.  By  heating  in  the  presence  of  alkali  and  completing  the 
reaction  by  the  addition  of  an  oxidizing  agent. 

4.  By  combustion  in  oxygen  in  a  closed  vessel. 

Since  the  choice  of  a  method  depends  on  the  nature  of  the 
sample  to  be  examined  and  the  apparatus  at  hand,  no  fixed 
rules  can  be  laid  down  for  this  purpose.  Several  of  the  more 
important  methods  are  therefore  outlined,  and  three  methods 
which  are  especially  useful  for  plant  and  animal  substances  are 
given  in  full. 

Of  methods  of  the  first  group,  that  of  Carius,  which  has  al- 
ready been  mentioned  in  the  previous  chapter,  is  the  most  im- 
portant. It  is  applicable  to  volatile  as  well  as  to  non-volatile 
substances,  but  is  somewhat  troublesome  and  tedious,  and  de- 
terminations are  frequently  lost  through  breaking  of  the  heated 
tubes,  especially  when  much  gas  is  produced  by  the  oxidation 
of  the  carbon  of  the  sample.  For  this  reason  only  a  small 
amount  of  any  highly  carbonaceous  substance  can  be  taken  for 
analysis,  and  often  the  amount  of  sulphuric  acid  obtained  is  too 
small  for  satisfactory  determination. 

Several  methods  belonging  to  the  second  group  have  been 
proposed.  Among  the  more  important  are  :  (a)  The  method 
of  Brugelmann,1  in  which  the  substance  is  burned  in  a  current 
of  oxygeri,  complete  oxidation  being  insured  by  passing  the 
mixture  of  gases  over  hot  platinum,  and  absorbing  the  sul- 
phuric acid  in  a  column  of  soda  lime.  If  the  substance  is  poor 
in  sulphur,  several  portions  can  be  burned  before  dissolving  out 
the  lime  and  determining  the  sulphuric  acid.  (5)  Mixter's 
modification  2  of  Sauer's  method.  This  consists  in  burning  the 
substance  in  a  current  of  oxygen  and  passing  the  products  of 
combustion  into  bromine  water.  (<?)  Classon's  method3  of 
burning  in  a  current  of  nitric  oxide,  (d)  Barlow's  method,4  in 
which  the  substance  is  burned  in  a  current  of  oxygen  in  a 
specially  constructed  tube  and  complete  oxidation  is  insured 
by  means  of  a  second  current  of  oxygen  introduced  laterally  at 

1  Z.  anal.  Chem.,  1876,  15,  1,  175 ;  1877,  16,  1. 

2  Am.  Chem.  J.,  1880,  2,  396.      8  Ber.,  1886,  19,  1910 ;  1887,  20,  3065. 
4  J.  Am.  Chem.  Soc.t  1904,  26,  341. 


NITROGEN,    SULPHUR,    AND    PHOSPHORUS  297 

a  point  between  the  boat  and  the  absorbing  column.  Methods 
of  this  type,  carefulty  carried  out,  insure  complete  oxidation, 
and  several  portions  of  a  sample  may  be  burned  in  succession 
so  as  to  secure  a  larger  amount  of  sulphuric  acid  for  determina- 
tion. They  are,  however,  less  rapid  than  the  methods  described 
below,  which  are  usually  quite  as  accurate. 

Liebig  destroyed  organic  matter  and  converted  sulphur  to 
sulphate  by  heating  with  a  mixture  of  caustic  alkali  and  alka- 
line nitrate,  and  methods  of  this  type  have  been  very  generally 
used  for  plant  and  animal  materials  and  other  non-volatile  sub- 
stances. Of  the  many  modifications  of  Liebig's  method,  the 
use  of  sodium  peroxide  as  an  oxidizing  agent  is  most  important. 
The  peroxide  may  be  used  in  connection  with  hydroxide  as  in 
Osborne's  method,  or  the  sample  may  be  mixed  at  once  with  an 
excess  of  peroxide  and  ignited  in  a  closed  crucible  1  or  in  the 
combustion  chamber  of  the  Parr  calorimeter.2 

Berthelot,  in  proposing  the  use  of  the  oxygen  calorimeter  for 
the  elementary  analysis  of  organic  materials,3  stated  that  on 
combustion  with  25  atmospheres  of  oxygen  the  sulphur  was 
completely  oxidized  and  in  the  presence  of  moisture  could  be 
quantitatively  recovered  as  sulphuric  acid.  This  method  has 
been  found  both  convenient  and  accurate.  Complete  oxidation 
takes  place  instantaneously,  arid  since  the  combustion  chamber 
is  hermetically  sealed  there  is  no  possibility  of  a  loss  of  volatile 
compounds.  A  small  autoclave  may  be  used  for  the  combustion 
if  a  bomb  calorimeter  is  not  available.  According  to  Hempel,4 
the  sample  can  be  burned  in  a  large  glass  bottle  filled  with 
oxygen  at  atmospheric  pressure. 

The  nature  and  number  of  the  samples  to  be  analyzed  will 
usually  determine  which  of  the  above  methods  is  to  be  pre- 

1Sundstrom:  J.  Am.  Chem.  Soc.,  1903,  25,  184.  Pennock  and  Morton: 
Ibid.,  25,  1265. 

2  Parr:    J.  Am.  Chem.  Soc.,    1900,  22,  646;   1904,  26,  1139.     Konek  :   Z. 
angew.  Chem.,  1903,  516.    Leclerc  and  Dubois :  J.  Am.  Chem.  Soc.,  1904,  26, 
1108. 

3  Compt.  rend.,  1892,  114,  318;    1899,  129,  1002.      See  also  Hempel:  Ber., 
1897,  30,  202,  and  LaDgbein  :  Z.  angew.  Chem.,  1900,  1227,  1259. 

4  Z.  angew.  Chem.,  1892,  393.     See  also  Graeffe :  Ibid.,  1904,  616. 


298  METHODS    OF    ORGANIC    ANALYSIS 

ferred.  For  the  majority  of  cases  either  Liebig's  or  Osborne's 
method,  or  if  the  facilities  are  available,  Berthelot's  method  of 
combustion  in  compressed  oxygen  may  be  recommended. 

LIEBIG'S  ALKALI  METHOD 

In  a  large  silver  or  nickel  crucible  mix  about  8  grams  potas- 
sium hydroxide  and  about  1  gram  potassium  nitrate.  Fuse  and 
stir  with  a  silver  or  nickel  spatula.  Allow  to  cool  and  add  1 
to  2  grams  of  the  finely  pulverized  sample.  Heat  gently,  and 
as  soon  as  the  mass  softens,  stir  well  so  as  to  bring  the  whole 
of  the  sample  into  contact  with  the  alkali  before  it  is  strongly 
heated,  then  gradually  increase  the  heat,  stirring  frequently  to 
keep  down  frothing,  and  continue  heating  until  the  mass  be- 
comes colorless.  For  samples  which  react  violently  when  stirred 
into  the  alkali  as  above,  a  smaller  proportion  of  nitrate  should 
be  used  at  the  beginning,  or  the  nitrate  may  be  omitted  at  the 
start  and  added  after  the  sample  has  been  mixed  with  the  fused 
hydroxide.  If  the  substance  is  not  quickly  oxidized  by  the 
fusion  mixture,  small  amounts  of  pulverized  nitrate  may  be 
added  from  time  to  time  and  the  heating  continued  with  fre- 
quent stirring.  The  fusion  will  usually  be  perfectly  colorless 
when  the  oxidation  of  organic  matter  is  complete.  After  cool- 
ing dissolve  the  fusion  in  water  in  a  beaker  or  casserole,  acidu- 
late with  hydrochloric  acid,  and  evaporate  to  dryness  to  expel 
nitric  and  nitrous  acids  and  heat  at  110°  to  dehydrate  silica  if 
present.  If  much  nitrate  or  silica  was  present,  the  evaporation 
with  hydrochloric  acid  should  be  repeated.  Dissolve  the 
residue  in  cold  water,  adding  a  few  drops  of  hydrochloric  acid, 
filter  if  not  perfectly  clear,  heat  the  solution  to  boiling,  and  pre- 
cipitate at  the  boiling  temperature  by  adding  a  solution  of 
barium  chloride,  drop  by  drop  with  constant  stirring,  until  in 
considerable  excess.  Boil  5  or  10  minutes  longer  and  allow  to 
stand  in  a  hot  place  until  the  precipitate  settles,  leaving  the 
supernatant  liquid  perfectly  clear.  Filter  and  wash  the  barium 
sulphate,  first  with  water  acidulated  with  hydrochloric  acid, 
then  with  water  till  free  from  chlorides,  and  finally  ignite  and 
weigh  with  the  usual  precautions. 


NITROGEN,    SULPHUR,    AND    PHOSPHORUS  299 

Notes  and  Precautions.  —  The  reagents  should  be  tested  by 
making  a  "  blank  "  determination  with  sugar  or  cellulose.  Do 
not  reject  this  blank  if  no  precipitate  appears  when  the  barium 
chloride  is  added,  but  allow  the  solution  to  stand  over  night  to 
insure  the  separation  of  any  small  amount  of  barium  sulphate 
present,  then  filter,  wash,  ignite,  and  weigh,  and  subtract  the 
weight  from  that  of  the  precipitate  found  in  the  determination. 
An  apparently  trifling  amount  of  precipitate  in  the  "  blank  "  may 
weigh  enough  to  appreciably  affect  the  results.  If  the  fusion 
is  heated  by  an  ordinary  gas  flame,  it  is  likely  to  absorb  sulphur 
compounds  from  the  latter.  To  avoid  this,  use  an  alcohol  flame 
for  heating  the  fusions.  It  cannot  be  expected  that  the  blank 
determination  should  show  accurately  the  sulphur  absorbed  from 
the  gas  flame.  Even  when  an  alcohol  flame  is  used  and  the 
sulphur  obtained  from  the  reagents  is  deducted,  the  results  are 
sometimes  too  high.  According  to  Keiser l  this  is  due  chiefly  to 
the  fact  that  the  fused  alkali  takes  up  some  silver  from  the 
crucible,  and,  on  acidifying  with  hydrochloric  acid,  the  silver 
chloride  formed  is  held  in  solution  by  the  excess  of  potassium 
chloride  present  and  is  afterward  carried  down  by  the  barium 
sulphate,  increasing  the  weight  of  the  precipitate.  Keiser  there- 
fore recommends  that  the  neutralized  solution  be  cooled,  diluted 
with  water  to  about  a  liter,  and  allowed  to  stand.  Any  silver 
chloride  present  will  then  precipitate  and  may  be  filtered  out  be- 
fore adding  barium  chloride. 

A  modification  which  is  often  useful  consists  in  heating  the 
substance  with  strong  nitric  acid  until  a  considerable  part  of  the 
organic  matter  has  been  oxidized,  after  which  an  excess  of  alkali 
is  added  and  the  mixture  transferred  to  a  crucible  and  carefully 
fused  until  oxidation  is  complete.  Hammarsten 2  found  this 
method  to  give  the  same  results  as  the  methods  of  Liebig  and  of. 
Classon  when  applied  to  casein,  egg  albumen,  and  gelatin. 

OSBORNE'S  PEROXIDE  METHOD  3 

Convert  about  10  grams  of  sodium  peroxide  into  hydroxide 
by  adding  to  it,  in  a  silver  or  nickel  crucible,  a  slight  excess  of 

lAm.  Chem.  J.,  1883,  5,  207.  2Z.  physiol.  Chem.,  1885,  9,  273. 

SJ.  Am.  Chem.  Soc.,  1902,  24,  142. 


300  METHODS    OF   ORGANIC    ANALYSIS 

water  and  heating  until  the  excess  is  boiled  off.  Allow  the  fu- 
sion to  cool  until  pasty,  then  add  one  to  two  grams  of  the  sample 
and  stir  it  into  the  alkali  as  quickly  as  possible.  Heat  gently 
and  stir  well  to  keep  down  the  frothing.  When  the  first  vigorous 
action  is  over,  heat  until  the  mass  fuses  and  stir  in  small  portions 
of  fresh  peroxids  until  decomposition  is  complete  and  the  fusion 
is  practically  colorless.  Then  allow  to  cool,  dissolve  the  fusion, 
and  acidulate  with  hydrochloric  acid;  boil  to  destroy  any  excess 
of  peroxide  and  to  expel  chlorine,  and  complete  the  determina- 
tion as  in  Liebig's  method. 

Notes.  —  Blank  determinations  must  be  made  as  in  the  case 
of  the  Liebig  method,  and  the  same  precautions  observed  to 
prevent  the  absorption  of  sulphur  compounds  from  gas  flames 
or  from  the  air  of  the  laboratory.  There  is  also  the  same  danger 
of  high  results  from  contamination  of  the  barium  sulphate  pre- 
cipitate. If  silica  is  absent,  the  acidulated  solution  need  not  be 
evaporated  to  dryness,  since  no  nitrate  is  used  in  the  fusion. 
For  all  such  cases  the  method  is  considerably  more  rapid  than 
that  of  Liebig,  though  requiring  somewhat  more  careful  attention 
during  the  fusion  of  the  substance  with  the  alkaline  oxidizing 
mixture.  This  method  has  been  extensively  used  during  recent 
years  in  the  analysis  of  proteins,  food  materials,  and  physiological 
products,  and  has  lately  been  adopted  by  the  Association  of 
Official  Agricultural  Chemists.  Several  methods  involving  the 
use  of  sodium  peroxide  had  previously  been  proposed,  of  which 
that  of  Asboth1  is  perhaps  the  best  known.  This  consists  in 
mixing  the  substance  with  10  grams  of  dry  sodium  carbonate 
and  5  grams  of  peroxide  in  a  nickel  crucible  and  heating  care- 
fully at  first  and  then  more  strongly  until  oxidation  is  complete. 
After  dissolving  the  fusion  in  water  the  solution  may  be  boiled 
with  bromine  to  insure  complete  oxidation.  The  alkaline  mix- 
ture here  used  being  much  less  fusible  than  sodium  or  potassium 
hydroxide,  the  method  should  be  used  only  for  such  substances 
as  do  not  readily  lose  sulphur  on  heating.  For  such  substances 
it  offers  the  advantage  of  simplicity  in  manipulation. 

A  modified  form  of  the  sodium  peroxide  method  is  recom- 
1  Chem.  Ztg.,  1895,  19,  2040. 


NITROGEN,    SULPHUR,    AND   PHOSPHORUS  301 

mended  by  Neumann  and  Meinertz:  Z.  physiol.   Chem.,  1904, 
43,  37.     See  also  Dubois:  J.  Am.  Ohem.  Soc.,  1905,  27,  729. 

A.    O.    A.    C.    METHOD1 

Place  from  1.5  to  2.5  grams  of  material  in  a  nickel  crucible 
of  about  100  cc.  capacity  and  moisten  with  approximately  2  cc. 
of  water.  Mix  thoroughly,  using  a  nickel  or  platinum  rod. 
Add  5  grams  of  pure  anhydrous  sodium  carbonate  and  mix. 
Add  pure  sodium  peroxide,  small  amounts  (approximately  0.50 
gram)  at  a  time,  thoroughly  mixing  the  charge  after  each  addi- 
tion. Continue  adding  the  peroxide  until  the  mixture  becomes 
nearly  dry  and  quite  granular,  requiring  usually  about  5  grams 
of  peroxide.  Place  the  crucible  over  a  low  alcohol  flame  (or 
other  flame  free  from  sulphur)  and  carefully  heat  with  occa- 
sional stirring  until  contents  are  fused.  (Should  the  material 
ignite,  the  determination  is  worthless.)  After  fusion  remove 
the  crucible,  allow  to  cool  somewhat,  and  cover  the  hardened 
mass  with  peroxide  to  a  depth  of  about  0.5  cm.  Heat  gradually, 
and  finally  with  full  flame  until  complete  fusion  takes  place, 
rotating  the  crucible  from  time  to  time  in  order  to  bring  any 
particles  adhering  to  the  sides  into  contact  with  the  oxidizing 
material.  Allow  to  remain  over  the  lamp  for  ten  minutes  after 
fusion  is  complete.  Cool  somewhat ;  place  warm  crucible  and 
contents  in  a  600-cc.  beaker  and  carefully  add  about  100  cc.  of 
water.  After  violent  action  has  ceased,  wash  material  out  of 
crucible,  make  slightly  acid  with  hydrochloric  acid  (adding  small 
portions  at  a  time),  and  complete  the  determination  by  precipi- 
tating with  barium  chloride  in  the  usual  manner. 

BERTHELOT'S  COMPRESSED  OXYGEN  METHOD 

Press  into  the  form  of  a  pellet  a  suitable  amount  of  the  sub- 
stance (usually  one  to  three  grams),  introduce  into  the  bomb 
calorimeter,  and  burn  in  oxygen  under  a  pressure  of  about  25 
atmospheres  in  the  same  manner  as  in  the  determination  of  heat 

1  Method  adopted  by  the  Association  of  Official  Agricultural  Chemists  for 
determination  of  sulphur  in  plants.  U.  S.  Dept.  Agr.,  Bur.  Chem.,  Bui.  107 
(Revised),  p.  23. 


302  METHODS   OF   ORGANIC   ANALYSIS 

of  combustion.1  Screw  into  the  exit  tube  of  the  bomb  a  coupling 
carrying  a  delivery  tube  of  about  0.5  mm.  internal  diameter 
and  connect  with  a  washing  cylinder  containing  a  little  water. 
Carefully  open  the  valve  and  allow  the  gas  to  bubble  through 
the  water  until  the  contents  of  the  bomb  reach  atmospheric 
pressure  ;  then  disconnect,  open  the  bomb,  and  rinse  out  all 
moisture  which  has  condensed  in  the  chamber,  on  the  lining  of 
the  cover,  or  on  the  rods  which  support  the  combustion  capsule. 
This  must  be  done  carefully,  as  the  rinsings  usually  contain  the 
greater  part  of  the  sulphur.  In  order  to  keep  down  the  volume 
of  the  solution,  the  water  from  the  washing  cylinder  may  be 
used  for  the  first  rinsing.  Dissolve  any  ash  found  in  the  com- 
bustion capsule  in  a  little  hydrochloric  acid,  remove  silica,  if 
necessary,  and  add  the  solution  to  that  obtained  by  rinsing  the 
bomb.  Examine  the  lead  gasket  in  the  cover  of  the  bomb,  and 
if  a  slight  film  of  sulphate  is  found,  wash  it  into  the  main  solu- 
tion.2 Finally  boil  this  solution  down  to  the  desired  volume, 
neutralize  any  excessive  amount  of  hydrochloric  acid  which  may 
have  been  introduced  with  the  solution  of  the  ash,  filter  if  nec- 
essary, and  determine  the  sulphuric  acid  by  precipitation  with 
barium  chloride  as  already  described. 

Notes.  —  This  method,  based  mainly  upon  the  suggestion  of 
Berthelot  and  the  subsequent  work  of  Hempel,  has  been  applied  3 
with  very  satisfactory  results  to  a  number  of  animal  and  vege- 
table substances  as  well  as  to  synthetic  organic  compounds 
containing  sulphur  in  forms  not  readily  oxidized  to  sulphuric 
acid  —  e.g.,  benzyl  sulphide  and  diphenyl-thiourea.  So  far  as 
tested,  no  cases  of  incomplete  combustion  have  been  found.  If 
this  were  suspected,  the  gas  drawn  off  after  the  combustion 
should  be  led  through  bromine  water  or  an  alkaline  bromine  solu- 
tion and  the  washings  of  the  bomb  added  to  this  solution  and 
boiled  to  insure  oxidation  of  sulphites  to  sulphates.  Unless 
the  bomb  is  emptied  gradually  in  some  such  way  as  has  been 

JAtwater  and  Snell:  J.  Am.  Chem.  8oc.,  1903,  25,  659. 

2  Such  minute  quantities  of  lead  sulphate  as  are  ordinarily  found  will  dissolve 
readily  in  the  solution  and  will  not  interfere  with  the  precipitation  of  sulphuric 
acid  as  barium  sulphate.  *J.  Am.  Chcm.  Soc.,  1902,  24.  1100. 


NITROGEN,    SULPHUR,    AND    PHOSPHORUS  303 

described,  there  is  danger  that  some  sulphuric  acid  may  be  car- 
ried away  mechanically  by  the  escaping  gas.  This  is  probably 
the  principal  cause  of  the  low  results  occasionally  reported. 
When  properly  carried  out  this  method  has  several  important 
advantages.  Combustion  takes  place  very  rapidly  and  with  no 
chance  for  the  escape  of  volatile  products.  The  products  of 
combustion  can  be  drawn  off  and  examined  as  desired.  The 
only  reagents  required  in  obtaining  the  sulphur  as  sulphuric 
acid  are  compressed  oxygen  and  hydrochloric  acid,  both  of 
which  are  easily  obtained  free  from  any  appreciable  amounts 
of  sulphur.  By  eliminating  the  use  of  alkali,  any  danger  of 
absorption  of  sulphur  compounds  from  the  air  is  avoided,  and 
no  considerable  amount  of  foreign  salts  is  introduced  into  the 
solution  from  which  the  sulphuric  acid  is  to  be  precipitated. 

THE  DETERMINATION  OF  PHOSPHORUS 

The  oxidation  of  organic  matter  for  the  determination  of 
phosphorus  may  be  accomplished  by  any  of  the  methods  de- 
scribed in  connection  with  the  sulphur  determination.  It  is  of 
course  unnecessary  in  this  case  to  avoid  the  use  of  gas  flames, 
and  with  most  substances  there  is  much  less  danger  of  loss  by 
volatilization  than  in  the  case  of  sulphur.  For  these  reasons 
and  because  of  the  care  required  in  dissolving  the  fused  residue 
of  phosphates  left  in  the  ignition  capsule,  the  compressed 
oxygen  method  has  no  such  marked  advantages  in  the  deter- 
mination of  phosphorus  as  in  that  of  sulphur. 

ALKALI  METHOD 

Liebig's  method  of  oxidation,  either  with  or  without  previ- 
ous digestion  with  nitric  acid  or  oxidation  by  peroxide,  may 
be  used,  but  the  use  of  sodium  carbonate,  as  in  the  following 
method,  is  generally  more  convenient. 

Mix  1  to  3  grams  substance  with  6  to  7  grams  dry  sodium 
carbonate  in  a  platinum  dish  and  spread  3  or  4  grams  of  the 
carbonate  over  the  mixture.  Heat  over  a  Bunsen  burner,  care- 
fully at  first  until  frothing  ceases,  then  strongly  until  the  mass 
fuses.  To  the  fusion  add  small  portions  of  pulverized  potas- 


304  METHODS    OR  ORGANIC   ANALYSIS 

slum  nitrate,  stirring  thoroughly  with  a  platinum  rod  or  spat- 
ula, until  the  mass  is  entirely  colorless.  The  whole  amount 
of  nitrate  required  does  not  usually  exceed  1  gram.  After 
cooling,  transfer  the  fusion  to  a  beaker,  dissolve  in  water,  add 
an  excess  of  nitric  acid,  and  boil.  Allow  the  solution  to  cool, 
neutralize  with  ammonia,  add  a  few  drops  of  nitric  acid  and 
then  a  moderate  excess  of  molybdate  solution.1  Digest  at 
about  65°  for  an  hour  with  occasional  shaking  or  stirring,  filter^ 
and  wash  with  cold  water  or  a  cold  solution  of  ammonium 
nitrate.  Test  the  filtrate  by  adding  more  molybdate  solution 
and  digesting  again  at  about  65°.  Place  the  beaker  used  for 
the  precipitation  under  the  funnel,  dissolve  the  precipitate 
through  the  paper  by  means  of  ammonia,  and  wash  thoroughly 
with  water,  keeping  the  volume  of  the  solution  below  100  cc. 
Nearly  neutralize  with  hydrochloric  acid,  cool  thoroughly,  and 
add  a  moderate  excess  of  magnesia  mixture,2  drop  by  drop,  with 
constant  stirring.  After  15  minutes  add  10  cc.  of  concentrated 
ammonia  or  its  equivalent  ;  let  stand  four  to  twenty-four 
hours  ;  filter  (preferably  on  an  asbestos  felt  in  a  Gooch  cruci- 
ble), wash  with  dilute  ammonia  (2.5  to  5  per  cent)  until  practi- 
cally free  from  chlorides,  ignite,  and  weigh  as  magnesium 
pyrophosphate. 

ACID    METHOD3 

To  1  to  3  grams  substance  in  a  Kjeldahl  flask  add  20  cc. 
concentrated  sulphuric  acid  and  10  grams  of  ammonium  nitrate, 
and  heat  carefully  until  frothing  ceases,  then  allow  to  cool 
somewhat,  add  10  grams  more  of  ammonium  nitrate,  and  heat 
to  boiling  ;  or  treat  the  sample  with  concentrated  or  fuming 
nitric  acid,  shaking  first  in  the  cold  then  warming  gently  till 
vigorous  action  ceases,  then  add  10  cc,  of  sulphuric  acid,  10 

1  Of  the  usual  molybdate  solution  containing  about  5  per  cent  of  molybdic 
acid,  add  at  least  50  cc.  for  every  decigram  of  phosphoric  acid  expected. 

2  If  the  magnesia  mixture  is  of  the  usual  strength,  containing  about  5  per  cent 
of  magnesium  chloride,  add  about  15  cc.  for  each  decigram  of  phosphoric  acid 
expected. 

3 Neumann:  Duboi*  Beymontfs  Archiv.  (physiol.  Abth.),  1897,  552;  1900, 
159;  Z.  physiol.  Chem.,  1900,  29,  146;  1902-3,  37,  115.  Sherman:  J.  Am. 
Chem.  Soc.,  1902,  24,  1100. 


NITROGEN,    SULPHUR,    AND   PHOSPHORUS  305 

grams  of  ammonium  nitrate,  heat  gently  till  frothing  ceases, 
and  then  boil.  Continue  boiling  until  the  liquid  in  the  flask 
is  concentrated  to  a  volume  not  greater  than  the  volume  of 
sulphuric  acid  which  has  been  used.  At  this  volume  the  boil- 
ing point  should  be  higher  than  that  of  sulphuric  acid  because 
of  the  ammonium  sulphate  in  the  solution.  Boil  at  this  higher 
temperature  for  about  half  an  hour  ;  then,  if  not  colorless,  cool 
somewhat,  add  nitric  acid  (concentrated  or  fuming)  and  boil 
again  until  the  nitric  acid  is  expelled  and  the  low  volume  and 
high  temperature  are  reached  again,  repeating  as  often  as  neces- 
sary until  the  liquid  is  colorless  and  shows  no  discoloration  when 
heated  for  half  an  hour  at  low  volume  and  high  temperature  as 
described.  Cool  the  colorless  solution,  wash  it  out  into  a  beaker, 
neutralize  with  ammonia,  add  15  to  20  grams  of  ammonium  ni- 
trate, and  determine  phosphoric  acid  as  in  the  preceding  method, 
adding  a  somewhat  greater  excess  of  the  molybdate  reagent. 

Notes.  —  This  method  has  been  found  to  give  good  results 
with  a  variety  of  foods  and  physiological  products.  The  large 
amount  of  sulphates  retards  the  formation  of  the  phosphomolyb- 
date  precipitate,  but  by  using  liberal  quantities  of  ammonium  ni- 
trate and  molybdate  reagent  and  allowing  at  least  an  hour  for  the 
precipitation,  the  whole  of  the  phosphoric  acid  is  obtained  with- 
out difficulty.  When  the  substance  is  first  treated  with  fuming 
nitric  acid,  a  smaller  quantity  of  sulphuric  acid  may  be  used  and 
the  phosphomolybdate  will  then  form  somewhat  more  quickly. 

In  applying  this  method  to  liquids  such  as  milk  or  urine,  a 
suitable  amount  of  sample  (25  cc.  milk  or  50  cc.  urine)  is  trans- 
ferred to  the  flask,  20  cc.  sulphuric  acid  added,  and  the  mixture 
heated  carefully  as  in  the  determination  of  nitrogen  until  the 
sample  is  well  charred  and  the  greater  part  of  the  water  has 
been  boiled  off.  Ammonium  nitrate  is  then  added  and  the  de- 
termination is  completed  as  above  described. 

MAGNESIUM  NITRATE  METHOD  l 

Mix  2  grams  of  the  dry  substance  (or  a  correspondingly  larger 
amount  if  a  liquid)  with  5  cc.  of  a  neutral  35  per  cent  solution 

1  Adopted  by  the  Association  of  Official  Agricultural  Chemists  for  the  deter- 
mination of  phosphorus  in  plant  substance. 


306  METHODS   OF   ORGANIC    ANALYSIS 

of  magnesium  nitrate  ;   dry  and  ignite ;  dissolve  the  residue  in 
acid  and  determine  phosphoric  acid  as  usual. 

This  method  is  convenient  but  has  not  been  so  thoroughly 
tested  on  different  organic  forms  of  phosphorus  as  have  the 
alkali  and  the  acid  methods  described  above. 

REFERENCES 
I 

ABDERHALDEN  :  Handbuch  der  Biochemischen  Arbeitsmethoden.     I. 

EPHRAIM  :  Original-arbeiten  iiber  Analyse  der  Nahrungsmittel. 

GATTERMANN  :  Practical  Methods  of  Organic  Chemistry. 

HOPPE-SEYLER  :  Fhysiologisch-  und  Pathologisch-Chemische  Analyse. 

KONIG  :  Chemie  der  Menschlichen  Nahrungs  und  Genussmittel.     III. 

LASSAR-COHN  :  Arbeitsmethoden  fur  Organise h-C he mische  Laboratorien. 

LEACH  :  Food  Inspection  and  Analysis. 

LINCOLN  and  WALTON  :  Quantitative  Analysis  for  Agricultural  Students. 

MEYER  :  Analyse  und  Konstitutionserrnittelung  organischer  Verbindungen. 

MORSE  :  Exercises  in  Quantitative  Chemistry. 

SUDBOROUGH  and  JAMES  :  Practical  Organic  Chemistry. 

U.  S.  Dept.  Agriculture,  Bureau  of  Chemistry,  Bui.  107  (Revised). 

VAUBEL  :  Quantitative  Bestimmung  organischer  Verbindungen. 

WEYL  :  Die  Methoden  der  Organischen  Chemie.     I. 

II 

1895.  DYER  :  (Applicability  of  the  Modified  Kjeldahl  Method).  J.  Chem. 
Soc.,  67,  811. 

1902.  SHERMAN  :  The  Determination  of  Sulphur  and  Phosphorus  in  Or- 
ganic Materials.  J.  Am.  Chem.  Soc.,  24,  1100. 

1904.  GIBSON  :  Study  of  the  Kjeldahl  Method.     J.  Am.  Chem.  Soc.,  26, 105. 
SHERMAN,  MCLAUGHLIN,  and  OSTERBERG:    The  Determination  of 

Nitrogen  in  Food  Materials  and  Physiological  Products.     J.  Am. 
Chem.  Soc.,  26,  367. 

SHERMAN  and  FALK:   The  Determination  of  Nitrogen  in  Organic 
Compounds.     /.  Am.  Chem.  Soc.,  .26,  1469. 

1905.  DUBOIS  :  Determination  of  Sulphur  and  Phosphoric  Acid  in  Foods, 

Feces,  and  Urine.     J.  Am.  Chem.  Soc.,  27,  729. 
FLAMAND  and  PRAGER  :  Modification  of  Kjeldahl  Method  for  Azo- 

compounds,  etc.     Ber.,  38,  558. 
MILBAUER  :  Determination  of  Nitrogen  in  Hydrazones  and  Osazones 

by  the  Kjeldahl  Method.     Z.  Zuckerind.  Bohmen,  28,  338 ;  Bio- 

chem.  Centrbl.,  1905,  469. 
SADTLER  :    Inner  Crucible  Method   for  Determining   Sulphur  and 

Halogens  in  Organic  Substances.    J.  Am.  Chem.  Soc.,  27,  1188. 


NITROGEN,    SULPHUR,    AND   PHOSPHORUS  307 

1906.  FOLIN:  On  Sulphate  and  Sulphur  Determinations.     J.  Biol.  Chem., 

1,  131. 

HAAS  :  Production  of  Methane  as  Source  of  Error  in  Nitrogen  Deter- 
minations by  the  Absolute  Method.  Proc.  Chem.  Soc.,  1906,  22, 
81;  Analyst,  31,  167. 

1907.  GLADDING:    Comparative  Work   on   Nitrogen  Estimations  by  the 

Kjeldahl  and  Gunning  Methods  and  by  a  Combination  of  the 
Two  Methods.  U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  105. 
p.  85. 

1908.  DENNSTEDT  :  Simultaneous  Determination  of  Nitrogen  and  Sulphur 

with  Carbon  and  Hydrogen.     Ber.,  41,  600,  2778. 
LEAVITT  and  LECLERC:    Loss  of  Phosphorus  in   Ashing   Cereals. 

J.  Am.  Chem.  Soc.,  30,  391,  617. 

RICHMOND:  Estimation  of  (Triazo)  Nitrogen.     Analyst,  33,  179. 
RUPP:  Determination  of  Sulphur  by  Carius  Method.     Chem.  Ztg., 

32,  984 ;  Chem.  Abs.,  3,  296. 

1909.  PENNY  :   Report  on  the  Determination  of  Nitrogen.     U.  S.  Dept. 

Agriculture,  Bur.  Chem.,  Bui.  122,  p.  85. 

TROWBRIDGE  :  Determination  of  Phosphorus  in  Flesh.  J.  Ind.  Eng. 
Chem.,  1,  675. 

1910.  ALLEN  and  JOHNSON  :  The  Exact  Determination  of  Sulphur  in  Sol- 

uble Sulphates.     J.  Am.  Chem.  Soc.,  32,  588. 

BROWN:  Note  on  Kjeldahl  Estimation  of  Nitrogen  in  Fatty  Sub- 
stances.    Chem.  News,  102,  51. 
GRINDLEY    and    Ross :    Determination    of  Inorganic    and   Organic 

Phosphorus  in  Meats.     J.  Biol.  Chem.,  8,  483. 
HIBBARD  :  Notes  on  the  Determination  of  Nitrogen  by  the  Kjeldahl 

Method.    /.  Ind.  Eng.  Chem.,  2,  463. 
JONES  and  KELLOGG:  Report  on  Nitrogen  Determinations.     U.  S. 

Dept.  Agriculture,  Bur.  Chem.,  Bui.  132,  p.  16. 
SCHREIBER:    Determination  of  Total   Sulphur  in   Organic  Matter. 

J.  Am.  Chem.  Soc.,  32,  977. 
TROWBRIDGE  and  STANLEY  :    Phosphorus  in  Flesh.     J.  Ind.  Eng. 

Chem.,  2,  212. 

1911.  Committee   Report   on   Determination   of  Nitrogen.     J.  Ind.  Eng. 

Chem.,  3,  691. 
VAN   RUN:    (Note    on    Technique    of    Nitrogen    Determinations). 

Pharm.  Weekbl,  48,  27;  Chem.  Abs.,  5,  2791. 
WATERS  and  TUTTLE  :  The  Determination  of  Total  Sulphur  in  India 

Rubber.     /.  Ind.  Eng.  Chem.,  3,  734. 
WHITTIER  :  Estimation  of  Inorganic  Phosphorus  in  Animal  Tissues. 

J.  Ind.  Eng.  Chem.,  3,  248. 


CHAPTER   XY 
Proteins  and  Proteases 

THE  proteins  are  nitrogen  compounds1  of  high  molecular 
weight  which  consist,  so  far  as  is  at  present  known,  essentially 
of  combinations  of  a-amino  acids  and  their  derivatives.  The 
classification  and  terminology  of  proteins  recommended  by  a 
joint  committee  of  the  American  Physiological  Society  and  the 
Society  of  Biological  Chemists  is  as  follows  : 

I.  Simple  Proteins.  —  Protein  substances  which  yield  only 
a-amino  acids  or  their  derivatives  on  hydrolysis. 

(a)  Albumins.'  —  Simple  proteins  soluble  in  pure  water  and 
coagulable  by  heat. 

(5)  Globulins.  —  Simple  proteins  insoluble  in  pure  water  but 
soluble  in  neutral  solutions  of  salts  of  strong  bases  with  strong 
acids. 

(<?)  Glutelins.  —  Simple  proteins  insoluble  in  all  neutral  sol- 
vents but  readily  soluble  in  very  dilute  acids  and  alkalies. 

(c?)  Alcohol-soluble  Proteins.  —  Simple  proteins  soluble  in 
relatively  strong  alcohol  (70-80  per  cent),  but  insoluble  in  water, 
absolute  alcohol,  and  other  neutral  solvents. 

(e)  Albuminoids.  —  Simple  proteins  which  possess  essentially 
the  same  chemical  structure  as  the  other  proteins,  but  are  char- 
acterized by  great  insolubility  in  all  neutral  solvents.  These 
form  the  principal  organic  constituents  of  the  skeletal  structure 
of  animals  and  also  their  external  coverings  and  its  appendages. 
This  definition  does  not  provide  for  gelatin,  which  is,  however, 
an  artificial  derivative  of  collagen. 

(/)  Histones.  —  Soluble  in  water  and  insoluble  in  very  dilute 
ammonia  and,  in  the  absence  of  ammonium  salts,  insoluble  even 
in  an  excess  of  ammonia ;  yield  precipitates  with  solutions  of 

1  Most  proteins  contain  between  15.5  and  18  per  cent  of  nitrogen.  On  the 
assumption  that  the  average  is  16  per  cent,  the  nitrogen  content  is  often  taken 
as  a  measure  of  the  amount  of  protein  present. 

308 


PROTEINS   AND    PROTEASES  309 

other  proteins  and  a  coagulum  on  heating  which  is  easily  soluble 
in  very  dilute  acids.  On  hydrolysis  they  yield  a  large  number 
of  amino  acids,  among  which  the  basic  ones  predominate. 

(#)  Protamines.  —  Simpler  polypeptids  than  the  proteins  in- 
cluded in  the  preceding  groups.  They  are  soluble  in  water, 
uncoagulable  by  heat,  have  the  property  of  precipitating  aqueous 
solutions  of  other  proteins,  possess  strong  basic  properties  and 
form  stable  salts  with  strong  mineral  acids.  They  yield  com- 
paratively few  amino  acids,  among  which  the  basic  amino  acids 
greatly  predominate. 

II.  Conjugated  Proteins.  —  Substances   which   contain   the 
protein  molecule  united  to  some  other  molecule  or  molecules 
otherwise  than  as  a  salt. 

(a)  Nucleoproteins.  —  Compounds  of  one  or  more  protein 
molecules  with  nucleic  acid. 

(5)  Gly  coproteins.  —  Compounds  of  the  protein  molecule  with 
a  substance  or  substances  containing  a  carbohydrate  group  other 
than  a  nucleic  acid. 

(<?)  Phosphoproteins.  —  Compounds  of  the  protein  molecule 
with  soiAe,  as  yet  undefined,  phosphorus- containing  substance 
other  than  a  nucleic  acid  or  lecithins. 

(d)  Hemoglobins.  —  Compounds  of  the  protein  molecule  with 
hematin  or  some  similar  substance. 

(e)  Lecithoproteins.  —  Compounds  of  the  protein  molecule 
with  lecithins  (lecithans,  phosphatids). 

III.  Derived  Proteins.  — 1.    Primary  Protein  Derivatives.  — 
Derivatives  of  the  protein  molecule  apparently  formed  through 
hydrolytic  changes  which  involve  only  slight  alterations  of  the 
protein  molecule. 

(a)  Proteans.  —  Insoluble  products  which  apparently  result 
from  the  incipient  action  of  water,  very  dilute  acids,  or  enzymes. 

(J)  Metaproteins.  —  Products  of  the  further  action  of  acids 
and  alkalies  whereby  the  molecule  is  so  far  altered  as  to  form 
products  soluble  in  very  weak  acids  and  alkalies,  but  insoluble 
in  neutral  fluids.  This  group  will  thus  include  the  familiar 
"acid  proteins"  and  "alkali  proteins,"  not  the  salts  of  proteins 
with  acids. 


310  METHODS   OF   ORGANIC    ANALYSIS 

(Y)  Coagulated  Proteins.  —  Insoluble  products  which  result 
from  (1)  the  action  of  heat  on  their  solutions,  or  (2)  the  action 
of  alcohols  on  the  protein. 

2.  Secondary  Protein  Derivatives.  —  Products  of  the  further 
hydrolytic  cleavage  of  the  protein  molecule. 

(a)  Proteoses.  —  Soluble  in  water,  uncoagulated  by  heat,  and 
precipitated  by  saturating  their  solutions  with  ammonium  sul- 
phate or  zinc  sulphate. 

(6)  Peptones.  —  Soluble  in  water,  uncoagulated  by  heat,  but 
not  precipitated  by  saturating  their  solutions  with  ammonium 
sulphate. 

(V)  Peptids.  —  Definitely  characterized  combinations  of  two 
or  more  amino  acids,  the  carboxyl  group  of  one  being  united 
with  the  amino  group  of  the  other,  with  the  elimination  of  a 
molecule  of  water. 

For  descriptive  accounts  of  the  proteins  reference  must  be 
made  to  some  of  the  works  cited  at  the  end  of  the  chapter.  In 
ordinary  analytical  work  some  of  the  groups  of  proteins  in- 
cluded in  the  above  classification  are  seldom  met  in  sufficient 
quantity  to  require  separate  consideration. 

The  groups  of  proteins  of  greatest  interest  to  the  analytical 
chemist  are  the  following  : 

Albumins.  —  Soluble  in  water  and  dilute  salt  solutions  ;  pre- 
cipitated by  adding  sufficient  alcohol  or  by  saturating  the  aque- 
ous solution  with  ammonium  sulphate.  The  water  solutions  are 
coagulated  by  heating,  commonly  at  70°-73°  (Halliburton),  but 
sometimes  much  lower,  leucosin  coagulating  at  52°  (Osborne). 

Globulins.  —  Insoluble  in  pure  water  but  soluble  in  dilute 
salt  solutions.  Precipitated  by  alcohol  or  by  saturation  with 
ammonium  or  magnesium  sulphate;  also  precipitated  from  salt 
solutions  on  removing  the  salt  by  dialysis.  Some  of  the  vege- 
table globulins  are  distinctly  crystalline  and  not  coagulable  by 
heating. 

Alcohol-soluble  Proteins.  —  These  occur  especially  in  the 
cereal  grains.  Gliadin,  insoluble  in  water,  dilute  salt  solution, 
or  absolute  alcohol,  but  soluble  in  75  per  cent  alcohol  is  the 
best  known  member  of  this  group. 


PROTEINS  AND  PROTEASES  311 

Albuminoids.  —  Some  of  these  differ  considerably  from  each 
other,  but  all  are  characterized  by  their  resistance  to  reagents. 
Among  the  more  important  of  the  albuminoids  are  collagens 
which  yield  gelatins  on  boiling  with  water ;  keratins  of  skin, 
horn,  hair,  feathers,  nails,  etc. ;  elastin  of  connective  tissue ; 
skeletins,  the  nitrogenous  compounds  of  the  skeletal  and  re- 
lated tissues  of  invertebrates,  including  the  characteristic  com- 
pounds of  sponges  and  silk. 

Gelatins  (and  collagens)  are  the  only  albuminoids  which  are 
likely  to  be  met  in  connection  with  the  true  proteins  in  foods, 
etc.,  so  that  the  other  substances  mentioned  need  not  be  con- 
sidered in  connection  with  the  reactions  given  below. 

Metaproteins.  —  By  the  action  of  acid  or  alkali  upon  native 
or  coagulated  protein  the  latter  may  be  converted  into  a 
metaprotein,  sometimes  called  an  acid-  or  an  alkali-albumin  or 
albuminate.  Both  acid  and  alkali  albuminates  are  nearly  in- 
soluble in  water  and  dilute  salt  solution,  but  usually  dissolve 
readily  in  water  containing  a  very  small  amount  of  acid  or 
alkali  and  are  precipitated  from  such  solutions  by  neutralization 
at  ordinary  temperature.  The  metaproteins  or  albuminates 
are  precipitated  from  nearly  neutral  solutions  by  the  general 
precipitants  for  soluble  proteins  (see  below)  but  a  very  minute 
amount  of  acid  or  alkali  is  sufficient  to  prevent  the  coagulation 
of  albuminate  by  boiling. 

Proteases  and  Peptones.  —  These  are  products  derived  from 
other  proteins  by  digestion  or  by  simple  hydrolysis.  They  are 
soluble  in  water  and  not  coagulated  by  boiling  their  aqueous 
solutions.  No  sharp  line  can  be  drawn  either  between  proteoses 
and  peptones  or  between  peptones  and  the  simpler  nitrogen 
compounds  which  result  from  prolonged  digestion.  As  the 
terms  are  generally  used,  peptones  may  be  considered  the  final 
products  of  digestion  or  hydrolysis  which  are  still  proteins  as 
judged  by  the  biuret  reaction  (see  below)  and  are  precipitable 
by  tannin  (perhaps  not  always  completely)  or  by  addition  of 
strong  alcohol.  Proteoses  (albumoses)  are  intermediate  products 
between  metaproteins  (albuminates)  and  peptones.  In  addition 
to  the  protein  reactions  shown  by  peptones,  the  proteoses  are 


312  METHODS   OF   ORGANIC   ANALYSIS 

precipitated  from  aqueous  solutions  at  ordinary  temperatures 
by  adding  acetic  acid  and  potassium  ferrocyanide  or  by  saturat- 
ing the  solution  with  zinc  or  ammonium  sulphate. 

The  term  peptone  was  formerly  applied  to  all  digestion  prod- 
ucts not  coagulated  by  boiling  and  is  still  popularly  used  in 
the  same  sense,  the  best  commercial  "peptones"  consisting 
largely  of  proteoses. 

Coagulated  Proteins. — This  group  includes  proteins  which 
have  been  coagulated  by  heating  or  by  alcohol.  The  nature  of  the 
change  which  takes  place  in  coagulation  is  not  known.  The  coag- 
ulated proteins  are  insoluble  in  water,  alcohol,  salt  solutions,  or 
very  dilute  acids.  They  are  dissolved  and  converted  into  meta- 
proteins  by  stronger  acids  and  alkalies,  especially  on  warming. 

G-lycoproteins.  —  Glyco-  or  gluco-proteins  are  compounds  of 
proteins  with  carbohydrates.  They  are  practically  insoluble 
in  water,  but  easily  soluble  in  very  weak  alkalies.  On  boiling 
with  dilute  mineral  acids  they  yield  considerable  amounts  of 
reducing  sugars.  Mucins  and  mucoids  belong  to  this  group. 

Nudeoproteins.  —  These  are  compounds  of  proteins  with 
nuclein  or  nucleic  acid.  Nudeoproteins  are  found  especially  in 
cell  nuclei  and  are  therefore  particularly  abundant  in  the 
highly  nucleated  cells  of  secreting  organs  such  as  the  liver, 
pancreas,  etc.,  and  in  the  germs  of  seeds. 

While  all  of  the  simple  proteins  are  levorotatory,  the  nucleo- 
proteins  thus  far  studied l  have  shown  dextrorotation  and  the 
nucleic  acid  of  the  wheat  embryo  has  been  found  to  be  strongly 
dextrorotatory.2 

Phosphoproteins. —  Casein  of  milk  and  vitellin  of  egg-yolk 
are  the  important  members  of  this  group.  Methods  for  the 
separation  of  casein  from  the  other  proteins  of  milk  are  in- 
cluded in  the  references  beyond. 

Other  Nitrogen  Compounds.  —  Among  the  organic  nitrogen 
compounds  which  occur  with  proteins  in  many  animal  or 
vegetable  substances  are  the  lecithins  and  related  compounds, 
alkaloids,  and  the  so-called  "nitrogenous  extractives,"  including' 
amines,  amids,  and  amino-acids.  Ammonium  salts  and  nitrates 

1  Gamgee  and  Jones  :  Am.  J.  Physiol.,  1903,  8,  447. 
20sborne  :  Ibid.,  1903,  9,  69. 


PROTEINS  AND  PROTEASES  313 

may  occur  in  plant  and  animal  tissues,  but  usually  only  in 
minute  amounts.  They  may,  however,  be  added  to  food 
materials  as  preservatives.  In  most  natural  food  products  the 
total  amount  of  these  simpler  nitrogen  compounds  is  small  as 
compared  with  that  of  proteins,  and  it  has  become  customary 
in  food  analysis  to  take  the  total  nitrogen  as  a  measure  of  the 
proteins  present,  so  that  the  percentage  of  proteins  as  reported 
ordinarily  means  the  percentage  of  nitrogen  multiplied  by  6.25, 
this  factor  being  based  on  the  assumption  that  proteins  contain 
approximately  16  per  cent  of  nitrogen. 

ANALYTICAL  REACTIONS  OF  PROTEINS 
Color  tests  and  precipitation  reactions  of  fairly  general  ap- 
plication to  proteins  are  known,  but  no  one  of  these  tests  or 
reactions  is  given  exclusively  by  proteins,  so  that  as  a  rule  in 
order  to  establish  the  presence  of  protein  beyond  doubt,  as 
many  as  possible  of  the  tests  should  be  applied.  Particularly 
in  examining  solutions  by  means  of  color  tests,  one  should  in 
important  cases  not  only  test  the  solution  but  also  obtain  the 
protein  from  the  solution  by  precipitation  arid  perform  the 
color  reaction  upon  this  precipitate.  For  this  purpose  it  is 
usually  best  to  precipitate  by  heat  coagulation  where  possible 
and  in  other  cases  to  precipitate  by  rendering  the  solution  slightly 
acid  with  acetic  acid  and  then  adding  a  solution  of  potassium 
ferrocyanide  which  under  these  conditions  gives  a  fine  white  pre- 
cipitate with  most  of  the  proteins  though  not  with  peptones  nor 
with  gelatin.  The  precipitate  thus  obtained  gives  color  reac- 
tions like  those  of  the  protein  itself.  (Winternitz  :  Z.  physiol. 
<7^w.,1892,i6,439.) 

COLOR   REACTIONS 

The  Biuret  Reaction l 

On  adding  a  very  dilute  solution  of  copper  sulphate  drop  by 
drop  to  a  protein  solution  strongly  alkaline  with  sodium  or 

iRose:  Poggendorff's  Annalen,  28,  132  (1833).  Piotrowski:  Sitzber.  Akad. 
d.  Wiss.  Wien,  math.-naturw.  Classe,  24,  335  (1875).  Wiedemann :  Poggen- 
dorff's  Annalen,  74,  67  (1849).  Pickering:  J.  Physiol.,  14,  347  (1803).  Schiff, 
Bcr.,  29,  298;  30,  2449;  LieUg's  Annalen,  299,  236;  303,  183;  307,  231; 
310,  37,  301 ;  319,  300. 


314          METHODS  OF  ORGANIC  ANALYSIS 

potassium  hydroxide  a  rose-red  to  violet  coloration  appears. 
A  more  pronounced  rose  color  is  obtained  with  proteoses  and 
peptones  than  with  other  proteins.  This  test  is  said  to  show 
1  part  of  peptone  in  100,000  of  solution.  For  other  proteins 
it  is  less  delicate.  To  test  for  peptones  in  a  solution  contain- 
ing other  proteins,1  precipitate  the  latter  by  saturation  with 
zinc  sulphate,  filter,  to  the  filtrate  add  caustic  soda  until  the 
zinc  hydroxide  first  precipitated  is  completely  dissolved,  then 
add  a  few  drops  of  a  one  per  cent  solution  of  copper  sulphate. 
The  reaction  is  given  by  many  substances  other  than  proteins  ; 
according  to  Schiff 2  by  any  compound  containing  two  CONH2 
groups  united  either  directly  by  their  carbon  atoms  or  by 
means  of  a  third  carbon  or  a  nitrogen  atom.  Examples  of 
these  three  classes  of  compounds  are  oxamid,  malonamid,  and 
biuret. 

CONH2  CONH2 

CONH2  /  / 

H2C  HN 

V.NH,  \0NH2 

Oxamid  Malonamid  Biuret 

For  cases  in  which  the  biuret  reaction  is  obtained  when  no 
—  CONH2  groups  appear  to  be  present,  as  well  as  for  a  general 
discussion  of  the  interpretation  of  the  test  in  work  upon  di- 
gestion and  synthetic  products,  see  Mann's  Chemistry  of  the 
Proteids,  pp.  141-144. 

According  to  Stokvis  (Z.  Biol.,  1896,  34,  466)  and  Sal- 
kowski  (^Berl.  Jclin.  Wochenschr.,  1897,  No.  17)  urobilin  gives 
under  the  same  treatment  a  color  which  cannot  be  distinguished 
from  that  of  the  biuret  reaction. 

Siegfried  has  also  noted  (Z.  physiol.  Chem.,  35,  164)  that 
the  biuret  reaction  may  be  obscured  by  the  presence  of  other 
bodies. 

1  Neumeister :  Z.  Biol.,  1890,  26,  324. 
>.,  1896,  29,  298. 


PROTEINS   AND    PROTEASES  315 

The  Xanthoproteic  Reaction  l 

Proteins  are  colored  yellow  by  nitric  acid  of  1.2  specific 
gravity  or  stronger.  The  color  is  intensified  by  heating  and 
changes  to  orange  or  red  on  treatment  with  an  excess  of  am- 
monia. According  to  Halliburton  2  this  reaction  depends  upon 
the  presence  of  an  oxybenzene  nucleus. 

Salkowski  has  proposed  (Z.  physiol.  Chem.,  12,  219  ;  Vaubel, 
I,  381)  to  make  use  of  this  reaction  for  the  approximate  colon- 
metric  estimation  of  peptones  in  solution. 

The  Millon  Reaction  3 

On  heating  with  Millon's  reagent4  (a  solution  of  mercuric 
nitrate  containing  nitrous  acid)  protein  matter  (including 
gelatin)  gives  a  brick-red  coloration  which  according  to  Nasse 
is  also  due  to  the  presence  of  any  oxybenzene  nucleus  in  the 
protein  molecule.  For  general  description  see  Mann,  p.  7,  and 
for  detailed  discussions  the  papers  of  Vaubel5  and  Nasse.6 

The  Tryptophan  Reaction9? 

When  protein  is  treated  first  with  glacial  acetic  acid  and  then 
with  concentrated  sulphuric  a  violet  color  usually  appears. 
Hopkins  and  Cole  8  found  that  the  reaction  occurred  only  when 
the  acetic  acid  contained  traces  of  glyoxylic  acid  and  that  a 
better  reaction  is  obtained  by  mixing  the  protein  with  a  little 

1  Fourcroy  and  Vauquelin  :  Ann.  Chim.,  56,  37.     Fiirth  :  Einwirkung  von  Sal- 
petersaure  auf  Eiweisstoffe,  Habilitationsschrift,  Straussburg,  1899.     Salkowski : 
Z.  physiol.  Chem.,  12,  215  (1887).     Rohde,  Ibid.,  44,  161  (1905). 

2  Schafer's  Textbook  of  Physiology,  I,  47. 

3  Millon:   Compt.  rend.,  28,  40  (1849). 

4  To  prepare  the  reagent  dissolve  mercury  in  twice  its  weight  of  nitric  acid, 
1.42  sp.  gr.,  and  dilute  the  solution  obtained  with  three  times  its  volume  of 
water.     According  to  Nasse  a  better  method  is  to  use  a  solution  of  mercuric 
acetate  containing  a  few  milligrams  of  sodium  or  potassium  nitrite,  the  solution 
having  been  recently  acidulated  with  acetic  acid. 

5  Z.  angew.  Chem.,  1900,  1125. 

6  Arch.  ges.  Physiol.,  (Pfluger),  1901,  83,  361. 

7  Adamkiewicz  :  Arch.  ges.  Physiol.,  9,  156  (1874)  and  Ber.,  8,  161  (1875). 

8  Proc.  Royal  Soc.,  1901,  68,  21 ;  J.  Physiol.,  27,  418  ;  29,  451. 


316  METHODS    OF    ORGANIC    ANALYSIS 

glyoxylic  acid  solution l  and  afterward  adding  concentrated 
sulphuric  acid.  The  color  then  appears  at  the  line  of  contact 
of  the  liquids.  This  reaction,  which  is  due  to  the  presence  of 
the  tryptophan  group  in  the  protein  molecule,  is  not  given  by 
gelatin.  For  other  color  reactions  due  to  tryptophan,  see  Cole  : 
J.  Physiol.,  1903,  30,  311. 

Several  other  more  or  less  characteristic  color  reactions  of 
proteins  may  be  found  by  consulting  the  references  given  at 
the  end  of  the  chapter. 

PROTEIN  PRECIPITANTS 

Heat  coagulation  and  salting  out  processes  have  already  been 
referred  to  in  the  general  description  of  the  proteins  and  have 
been  made  use  of  in  classification. 

Zinc  sulphate  is  most  used  for  salting-out  in  analytical  work 
where  the  amount  of  protein  precipitated  is  to  be  found  by 
determining  nitrogen  in  the  precipitate. 

Heavy  metals  have  been  considerably  used  as  precipitants. 
Among  these  may  be  mentioned  iron  as  chloride  or  acetate, 
copper  as  sulphate,  acetate,  or  hydroxide,  lead  as  neutral  or  basic 
acetate,  mercury  as  chloride  or  as  mercury-potassium-iodide, 
uranium  as  acetate. 

Halogens  form  insoluble  or  sparingly  soluble  compounds  with 
proteins,  and  bromine  especially  has  been  used  as  precipitant  in 
analytical  work. 

Among  the  acids  which  are  good  precipitants  for  both  proteins 
and  alkaloids  are  phosphotungstic  and  phosphomolybdic  acids, 
tannic  acid,  picric  and  picrolonic  acids,  trichlor-acetic  acid. 

Zinc  sulphate?  —  In  general  on  saturating  a  solution  with  zinc 
sulphate  all  proteins  present  except  peptones  are  precipitated. 
This  precipitation  may  therefore  be  used  to  separate  peptones 

1  Prepared  as  follows  :  Place  a  saturated  solution  of  oxalic  acid  in  a  tall 
cylinder,  add  lumps  of  sodium  amalgam  (about  60  grams  per  liter  of  solution), 
allow  to  stand  as  long  as  hydrogen  is  evolved,  then  filter  and  dilute  the  solution 
with  twice  its  volume  of  water. 

2  Bonier:  Z.  anal.  Chem.,  1895,  34,  562.    Baumann  and  Bonier  :  Nahr.Zen- 
ussm.,  1898,  1,  106.     Zunz  :  Z.  physiol.  Chem.,  1899,  27,  217.     Van  Slyke  and 
Hart :  Am.  Chem.  J.,  1903,  29,  150. 


PROTEINS   AND    PROTEASES  317 

from  other  proteins.  It  is  especially  employed  in  the  analysis 
of  mixtures  of  proteins  in  connection  with  artificial  digestion 
experiments.  Here  the  proteins  other  than  proteoses  and  pep- 
tones can  usually  be  removed  in  other  ways  (for  instance  by 
neutralizing  and  heating  the  solution),  the  proteoses  separated 
by  saturating  the  solution  with  zinc  sulphate,  and  the  peptones 
determined  in  the  filtrate. 

•  Ferric  acetate  is  recommended  by  Allen 1  as  an  efficient  pre- 
cipitant. A  neutral  solution  of  the  reagent  is  added  in  excess 
and  the  liquid  rapidly  boiled,  when  all  protein  will  be  precipi- 
tated and  can  be  determined  from  the  amount  of  nitrogen  found 
in  the  precipitate  by  the  Kjeldahl  method. 

Copper2  is  probably  more  generally  used  than  any  of  the  other 
heavy  metals  for  the  analytical  precipitation  of  proteins,  espe- 
cially for  the  separation  of  protein  from  non-protein  nitrogen 
in  vegetable  products.  Moist  cupric  hydroxide  (often  called 
Stutzer's  reagent)  is  most  commonly  used. 

This  method  has  been  adopted  by  the  Association  of  Official 
Agricultural  Chemists  in  the  following  form : 

Separation  of  Proteins  from  Amids,  etc. 

Preparation  of  Reagent.  — Dissolve  100  grams  of  pure  cupric 
sulphate  in  5  liters  of  water,  add  25  cc.  of  glycerol,  and  then  a 
dilute  solution  of  sodium  hydroxide  until  the  liquid  is  alkaline; 
filter;  rub  the  precipitate  up  with  water  containing  5  cc.  of 
glycerol  per  liter,  and  wash  by  decantation  or  filtration  until 
the  washings  are  no  longer  alkaline.  Rub  the  precipitate  up 
again  in  a  mortar  with  water  containing  10  per  cent  of  glycerol, 
thus  preparing  a  uniform  gelatinous  mass  that  can  be  measured 
out  with  a  pipette.  Determine  the  quantity  of  cupric  hy- 
droxide per  cubic  centimeter  of  this  mixture. 

.Determination.  —  Place  0.7  gram  of  the  substance  in  a  beaker, 
add  100  cc.  of  water,  heat  to  boiling,  or,  in  the  case  of  sub- 

1  Commercial  Organic  Analysis,  IV  (2),  38  (1898);  and  Vaubel:  Bestimmung 
organischer  Verbindungen,  I,  227  (1902). 

2  Stutzer,  J.  Landw.,  1880,  28, 103  ;  1881,  29,  473  ;  Z.  anal  Chem.,  1895,  34, 
568.     Mallet,  U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  54.     Fraps  and  Bizzell, 
J.  Am.  Chem.  Soc.,  22,  709. 


318  METHODS   OF    ORGANIC   ANALYSIS 

stances  rich  in  starch,  heat  on  the  water  bath  ten  minutes;  add 
a  quantity  of  cupric  hydroxide  mixture  containing  about  0.5 
gram  of  the  hydroxide,  stir  thoroughly,  filter  when  cold,  wash 
with  cold  water,  and,  without  removing  the  precipitate  from 
the  filter,  determine  nitrogen  by  the  Kjeldahl  method,  adding 
sufficient  potassium  sulphide  solution  to  completely  precipitate 
all  copper  and  mercury.  The  filter  papers  used  must  be  practi- 
cally free  from  nitrogen.  If  the  substance  examined  consists  of 
seed  of  any  kind,  or  residues  of  seeds,  such  as  oil  cake  or  anything 
else  rich  in  alkaline  phosphates,  add  a  few  cubic  centimeters  of 
a  concentrated  solution  of  alum  just  before  adding  the  cupric 
hydroxide,  and  mix  well  by  stirring.  This  serves  to  decom- 
pose the  alkaline  phosphates.  If  this  be  not  done,  cupric  phos- 
phate and  free  alkali  may  be  formed,  and  the  protein  copper 
precipitate  may  be  partially  dissolved  in  the  alkaline  liquid. 

This  method  has  been  considerably  criticized,  and  to  some 
extent  replaced  by  precipitation  with  other  reagents,  especially 
phosphotungstic  acid,  but  Fraps  and  Bizzell  consider  it  still  the 
best  method  available  for  vegetable  materials.  According  to 
Schjerning's  work  it  is  likely  to  precipitate  a  considerable  part 
of  any  purin  body  which  may  be  present. 

Lead l  is  an  effective  precipitant  of  proteins  as  shown  by  its 
successful  use  as  a  "  clarifier  "  in  preparing  liquids  containing 
sugar  for  polariscopic  examination,  for  all  proteins  are  optically 
active,  and  if  not  completely  removed  from  the  solution,  would 
vitiate  the  polariscopic  determination  of  sugar. 

Mercuric  chloride 2  precipitates  the  proteins,  including  pep- 
tones, but  is  also  liable  to  give  precipitates  with  alkaloids  and 
even  with  ammonium  salts. 

Uranium  acetate  precipitates  proteins,  and  according  to 
Schjerning's  experiments  3  does  not  precipitate  the  alkaloids  or 
simpler  nitrogen  compounds  likely  to  be  met  in  analytical  work, 
with  the  exception  of  piperazine. 

1  Hofmeister:  Z.  physiol.  Chem.,  1878,  2,  288. 

2  Kuhne  :  Z.  Biol.,  1885,  22,  423.    Neumeister  :  Z.  Biol.,  1890,  26,  234.     Sieg- 
fried: Z.  physiol.  Chem.,  1902,35,  164. 

8  Summarized  by  Vaubel :  Bestimmung  organischer  Verbindungen,  I,  224- 
232. 


PROTEINS  AND  PROTEASES  319 

According  to  Schultz  and  Barbieri  as  quoted  by  Allen,  the 
greatest  source  of  error  in  the  quantitative  separation  of  pro- 
teins from  simpler  nitrogen  compounds  by  means  of  reagents 
such  as  the  above  lies  in  the  precipitation  of  more  or  less  of  the 
other  nitrogen  compounds  along  with  the  proteins.  Hence  they 
recommend  that  parallel  precipitations  be  made  with  several 
different  reagents.  If  then  all  the  nitrates  be  proven  free  from 
protein  by  testing  with  acetic  acid  and  potassium  ferrocyanide, 
and  the  nitrogen*  be  determined  in  each  precipitate,  the  lowest 
result  found  is  presumably  the  most  nearly  correct. 

Bromine  precipitation  is  sometimes  employed  for  separation  of 
proteins  from  simpler  nitrogen  compounds,  such  as  the  "  ex- 
tractives "  of  meat.  The  following  example  is  from  the  meth- 
ods of  the  Association  of  Official  Agricultural  Chemists: 

Separation  of  Proteoses,  Peptones,  and  Gelatin  from  Extractives 

This  is  a  combination  of  Bomer's 1  method  with  that  of  Allen 
and  Searle,2  as  modified  by  Wiley.3 

Evaporate  the  filtrate  from 'the  globulins  to  small  volume,  add 
2  or  3  drops  of  (1  : 3)  sulphuric  acid,  and  saturate  with  pow- 
dered zinc  sulphate.  The  excess  of  zinc  sulphate  added  should 
not  be  large,  as  otherwise  serious  "  bumping  "  is  likely  to  ensue. 
About  80  grams  of  the  salt  are  required  for  each  50  cc.  of 
liquid.  Allow  the  coagulated  proteins  to  settle,  filter,  and  wash 
with  a  saturated  solution  of  zinc  sulphate. 

Acidulate  the  filtrate  from  the  zinc  sulphate  precipitate  with 
2  or  3  drops  of  strong  hydrochloric  acid,  dilute  with  an  equal 
volume  of  water,  add  about  2  cc.  of  liquid  bromine,  and  shake 
the  contents  of  the  flask  vigorously.  (This  can  be  most  con- 
veniently done  in  a  Kjeldahl  flask.)  If  the  bromine  be  all  taken 
up,  add  more  until  about  0.5  cc.  of  liquid  bromine  is  left  undis- 
solved  and  the  supernatant  liquid  thoroughly  saturated.  Allow 
the  mixture  to  stand  over  night,  decant  the  supernatant  liquid 
through  a  filter  paper,  and  wash  with  water,  so  directing  the  jet 
that  the  globule  of  bromine  is  stirred  up  and  saturates  the  wash 

1  Z.  anal.  Chem.,  1895,  5,  562. 

2  Analyst,  1897,  22,  258-263. 

3  U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  54. 


320  METHODS   OF   ORGANIC    ANALYSIS 

water.  Return  the  filter  paper  and  precipitate  to  the  flask,  add 
the  zinc  sulphate  precipitate  and  filter  paper  containing  it,  and 
determine  the  nitrogen.  The  percentage  of  nitrogen  so  found, 
multiplied  by  6.25,  gives  the  percentage  of  proteoses,  peptones, 
and  gelatin,  including  gelatin  peptone. 

Alkaloids,  if  present,  are  likely  to  be  more  or  less  completely 
precipitated  by  bromine  along  with  the  protein  matter. 

Phosphotungstic  acid  is  an  efficient  precipitant  of  the  proteins 
other  than  peptones.  At  ordinary  temperatures  simpler  nitro- 
gen compounds  are  also  thrown  down,  but  according  to  Mallet 
(Bui.  54,  Div.  Chem.)  a  satisfactory  separation  of  ordinary 
proteins  from  the  usual  nitrogenous  extractives,  such  as  occur 
in  meat  extract,  may  be  effected  by  precipitating  at  90°  and 
washing  with  water  at  the  same  temperature.  Fraps  and  Biz- 
zell  (J.  Am.  Chem.  Soc.,  22,  709)  question  the  completeness  of 
precipitation  of  proteins  at  temperatures  above  60°.  Alkaloids 
if  present  would  be  likely  to  be  precipitated  about  as  com- 
pletely as  the  proteins. 

Stutzer  (Z.  anal.  Chem.,  1895,  34,  568;  1896,  35,  493)  and 
also  Bondzynski  (Landw.  Jahrb.  der  Schweiz,  1894)  used  phos- 
photungstic  acid  as  precipitant  to  separate  proteins,  including 
peptones,  from  amino-compounds.  Van  Slyke  and  Hart  (Bui. 
215,  New  York  Agricultural  Experiment  Station,  Geneva)  con- 
sider that  precipitation  with  this  reagent  in  the  cold  gives  a 
satisfactory  separation  of  the  proteins  of  cheese  from  mono- 
amino  compounds,  but  is  liable  to  precipitate  di-amino-com- 
pounds  if  these  are  present,  arginin  giving  a  precipitate  with 
phosphotungstic  acid  which  is  soluble  when  hot,  but  separates 
out  on  cooling,  while  the  lysin,  histidin,  and  putrescine  precip- 
itates failed  to  redissolve  completely  even  on  boiling. 

In  using  phosphotungstic  acid  for  the  determination  of  pro- 
tein nitrogen  in  meat  extract  according  to  the  suggestion  of 
Mallet,  the  solution  is  first  acidulated  with  acetic  acid  and 
boiled,  filtered  if  necessary,  and  then  treated  with  a  slight  ex- 
cess of  a  solution  of  phosphotungstic  acid  in  2.5  per  cent  hydro- 
chloric acid,  heated  to  90°,  filtered  hot,  and  the  precipitate 
washed  with  water  at  about  the  same  temperature.  The  re- 


PROTEINS   AND    PROTEASES  321 

suits  thus  obtained  are  usually  slightly  higher  and  somewhat 
more  uniform  than  those  obtained  by  precipitation  with 
bromine. 

Tannin l  precipitates  proteins,  including  peptones,  from  solu- 
tions acidulated  with  acetic  acid,  the  precipitation  being  favored 
by  the  presence  of  sodium  chloride.  According  to  Allen  and 
also  Halliburton,  this  precipitation,  even  in  the  case  of  pep- 
tones, is  complete.  Van  Slyke  and  Hart,  however,  found  that 
tannin  precipitated  much  less  of  the  nitrogen  compounds 
formed  by  peptic  digestion  of  cheese  than  was  precipitated  by 
phosphotungstic  acid ;  and  after  investigation  they  attributed 
this  discrepancy  mainly  to  incomplete  precipitation  of  peptones 
by  tannin.  They  concluded  that  phosphotungstic  acid  should 
be  used  to  separate  peptones  from  amino  bodies  when  the 
amount  of  the  latter  is  relatively  small  or  when  they  consist 
largely  of  mono-amino-compounds  ;  while  tannin  should  be  pre- 
ferred as  a  precipitant  in  those  cases  in  which  the  amino-acids 
are  present  in  relatively  large  amount  as  compared  with  pep- 
tones or  when  considerable  amounts  of  di-amino-compounds  are 
present. 

Bigelow  and  Cook  (J".  Am.  Chem.  Soc.,  1906,  28,  1485)  as 
the  result  of  a  detailed  study  recommend,  for  the  separation  of 
proteoses  and  peptones  from  the  amino  bodies,  the  use  of  liberal 
amounts  of  tannin  and  salt  at  a  temperature  of  approximately 
12°  C.  Their  method  is  essentially  as  follows  : 

Dissolve  1  gram  of  the  sample  if  a  powder,  2  grams  if  a 
paste,  or  10  grams  if  a  concentrated  solution,  in  a  little  cold 
water. in  a  100-cc.  flask,  keeping  the  volume  within  20  cc.  [Or 
measure  into  the  flask  20  cc.  of  the  sample  if  a  dilute  solution.] 
Add  50  cc.  of  a  solution  containing  15  grams  of  sodium  chlo- 
ride, shake  well,  cool  to  approximately  12°,  add  30  cc.  of  a 
24  per  cent  tannin  solution  at  the  same  temperature,  dilute 
with  cold  water  to  100  cc.,  shake  thoroughly  and  allow  to  stand 
over  night  in  an  ice  box  at  about  12° ;  then  filter  at  the  same 
temperature  and  determine  nitrogen  both  in  an  aliquot  part  of 

1  Allen  :  Commercial  Organic  Analysis,  Vol.  IY  (2d.  ed.),  p.  19.  Schjerning  : 
Z.  anal.  Chem.,  1900,  39,  545. 


322          METHODS  OF  ORGANIC  ANALYSIS 

the  filtrate  and  in  a  similar  portion  of  the  nitrate  from  a  blank 
in  which  the  reagents  alone  have  been  used.  The  corrected 
nitrogen  of  the  nitrate  represents  that  which  was  present  as 
ammonia  and  amino  compounds  except  that  creatin  if  present 
is  partly  precipitated  by  the  tannin-salt  reagent.  The  amount 
of  creatin  thus  thrown  down  may  be  found  by  estimating  the 
amounts  in  the  original  solution  and  in  the  nitrate  colorimetri- 
cally  by  the  method  of  Folin,  the  excess  of  tannin,  being  re- 
moved from  the  filtrate  by  means  of  barium. 

Picric  acid  precipitates  proteins  very  completely  from  solu- 
tions acidulated  with  acetic  acid,  but  as  the  reagent  contains 
nitrogen  its  use  in  quantitative  analytical  work  is  limited. 
Gelatin  is  precipitated  when  the  picric  acid  is  in  excess,  and 
milk  or  cream  may  be  tested  for  gelatin  by  removing  the  milk 
proteins  with  acid  mercuric  nitrate  and  adding  to  the  clear 
filtrate  an  excess  of  a  saturated  solution  of  picric  acid,  when 
gelatin,  if  present,  yields  a  yellow  precipitate. 

Trichlor  acetic  acid  appears  to  be  a  convenient  reagent  for 
the  separation  of  proteins  under  certain  conditions,  but  has  not 
been  much  used  in  analytical  work.  According  to  Halliburton  * 
the  protein  solution  is  mixed  with  an  equal  volume  of  a  10  per 
cent  solution  of  trichloracetic  acid,  boiled  and  filtered  hot, 
when  the  filtrate  will  contain  the  proteoses  and  peptones  while 
the  other  proteins  are  precipitated.  In  the  cold  the  proteoses 
are  partially  precipitated  (Martin). 

SEPARATION  OF  PROTEINS  FROM  SIMPLER  NITROGEN  COM- 
POUNDS AND  FROM  EACH  OTHER 

The  separation  of  proteins  from  simpler  nitrogen  compounds 
is  usually  based  upon  the  precipitation  of  the  former  by  some 
one  of  the  above  reagents  which  will  not  precipitate  the  other 
nitrogen  compounds  believed  to  be  present  in  the  substance 
under  examination.  The  choice  of  precipitant  will  therefore 
depend  upon  the  nature  of  the  substance  from  which  the  pro- 

1  Schafer's  Physiology,  I.  40-41,  where  the  following  references  are  given: 
Obermayer,  Med.  Jalirb.,  Wien,  1888,  375;  Starling,  J.  PhysioL,  14,  131; 
Martin,  same,  15,  375 ;  Halliburton,  same,  17,  169,  and  J.  Path,  and  Bact., 
3,  295.  See  also  Mendel  and  Blood,  J.  Biol.  Chem.,  8,  186,  189. 


PROTEINS   AND   PROTEASES  323 

tein  is  to  be  separated.  Cupric  hydroxide  used  as  described 
above  has  been  most  commonly  employed  for  vegetable  mate- 
rials, while  precipitation  with  phosphotungstic  acid,  bromine 
or  tannin  and  salt  as  described  in  the  foregoing  paragraphs 
has  been  more  commonly  used  for  animal  substances.  The 
methods  of  separating  proteins  from  each  other  are  based 
almost  entirely  upon  differences  in  solubilities  and  coagulation 
or  other  precipitation  reactions  such  as  have  been  given  in 
characterizing  the  groups  already  mentioned.  As  the  reactions 
of  the  peptones  approach  those  of  some  of  the  compounds  of 
known  structure,  the  separation  of  proteins  from  simpler  com- 
pounds and  from  each  other  can  best  be  studied  as  parts  of  the 
same  problem.  The  methods  available  for  these  separations 
are  so  dependent  upon  the  particular  combination  of  com- 
pounds to  be  separated  that  any  attempt  to  give  detailed  direc- 
tions would  be  of  less  value  than  references  to  the  original 
publications,  a  list  of  which  will  be  found  at  the  end  of  this 
chapter. 

PROTEASES  OR  PROTEOLYTIC  ENZYMES 

For  the  determination  of  proteolytic  powers  of  substances 
containing  proteases  (pepsin,  trypsin,  papain,  etc.)  many  differ- 
ent methods  have  been  used.  Some  of  these  may  be  grouped  as 
follows  : 

1.  The  enzyme  acts  upon  an  insoluble  protein  and  the  rate 
at  which  the  latter  is  digested  into  soluble  products  is  observed 
(methods  of  Bidder  and  Schmidt,  Griinhagen,  Grutzner,  Mett, 
Palladin,  U.  S.  Pharmacopoeia). 

2.  The  enzyme  acts  upon  a  solution  or  suspension  of  protein 
and  the  time  required  to  carry  the  digestion  to  a  definite  stage, 
or  the  amount  of  protein  remaining  undigested  at  the  end  of  a 
definite  time,  is  determined  (methods  of  Allen,  Einhorn,  Fuld 
and  Levison,  Gross,  Jacoby-Solms,  Robertson,  Rose,   Thomas 
and  Weber,  U.  S.  Pharmacopoeia,  Witte). 

3.  The  enzyme  acts  upon  protein  or  polypeptid,  and   the 
cleavage  products  are  determined  by  chemical  or  physical  meth- 
ods  (methods  of  Abderhalden,  Allen,  Hedin,  Kober,  Koelker, 
Levene  and  Rouiller,  Schiff,  Schiitz,  Sorenson,  Volhard). 


324          METHODS  OF  ORGANIC  ANALYSIS 

4.  The  enzyme  is  allowed  to  act  on  a  protein  solution,  and 
the  progress  of  the  digestion  is  measured  by  increase  of  electri- 
cal conductivity  or  decrease  of  turbidity  or  viscosity  of  the  solu- 
tion (methods  of  Bayliss,  Hata,  Liebermann,  Spriggs). 

Since  the  newer  methods,  such  as  the  copper  method  of  Kober 
and  the  optical  method  of  Abderhalden  and  Koelker,  are  still 
being  developed  in  detail,  it  seems  best  for  the  purposes  of  this 
work  to  describe  fully  only  a  few  of  the  older  methods  which 
are  now  in  more  general  use,  after  which  will  be  given  a  chrono- 
logical list  of  references  which  will  guide  the  reader  to  the 
literature  of  the  more  recent  methods. 

U.  S.  PHARMACOPOEIA  METHOD  FOR  PEPTIC  ACTIVITY 

Mix  9  cc.  of  hydrochloric  acid  of  10  per  cent  strength  by 
weight  (1.049  sp.  gr.  at  25°  C.)  with  291  cc.  of  distilled  water 
and  dissolve  0.100  gram  of  the  pepsin  to  be  tested  in  150  cc.  of 
the  acid  liquid.  Immerse  a  hen's  egg,  which  should  be  fresh, 
during  15  minutes  in  boiling  water  ;  remove  the  pellicle  and  all 
of  the  yolk  ;  rub  the  white  coagulated  albumin  through  a  clean 
No.  40  sieve.  Reject  the  first  portion  that  passes  through  the 
sieve,  and  place  10  grams  of  the  succeeding  portion  in  a  wide- 
mouthed  bottle  of  100  cc.  capacity.  Add  20  cc.  of  the  acid 
liquid  and,  with  the  aid  of  a  glass  rod  tipped  with  cork  or  black 
rubber  tubing,  completely  disintegrate  the  albumin  ;  then  rinse 
the  rod  with  15  cc.  more  of  the  acid  liquid  and  add  5  cc.  of  the 
solution  of  pepsin.  Cork  the  bottle  securely,  invert  it  three 
times,  and  place  it  in  a  water  bath  that  has  previously  been 
regulated  to  maintain  a  temperature  of  52°  C.  Keep  at  this 
temperature  for  2^  hours,  shaking  every  10  minutes  by  invert- 
ing the  bottle  once.  Then  remove  it  from  the  water  bath,  add 
50  cc.  of  cold  distilled  water,  transfer  the  mixture  to  a  narrow 
graduated  cylinder,  and  allow  it  to  stand  for  half  an  hour.  The 
deposit  of  undissolved  albumin  should  not  then  measure  more 
than  1  cc. 

The  relative  proteolytic  power  of  pepsin  stronger  or  weaker 
than  that  just  described  may  be  determined  by  ascertaining 
through  repeated  trials  the  quantity  of  the  above  pepsin  solu- 


PROTEINS   AND    PROTEASES  325 

tion  required  to  digest,  under  the  prescribed  conditions,  10  grams 
of  boiled  and  disintegrated  egg  albumin.  Divide  15,000  by  this 
quantity  expressed  in  cubic  centimeters  to  ascertain  how  many 
parts  of  egg  albumin  one  part  of  pepsin  will  digest. 

U.  S.  PHARMACOPOEIA  TEST  FOR  TRYPTIC  ACTIVITY  OF 
PANCREATIN 

If  0.28  gram  of  pancreatin  and  1.5  grams  of  sodium  bicar- 
bonate be  added  to  100  cc.  of  tepid  water  contained  in  a  flask, 
and  if  400  cc.  of  fresh  cows'  milk,  which  has  been  previously 
heated  to  38°  C.,  be  then  added,  and  the  temperature  of  the 
mixture  maintained  at  this  point  for  30  minutes,  the  milk 
should  be  so  completely  peptonized  that,  if  a  small  portion  of 
it  be  transferred  to  a  test  tube  and  mixed  with  some  nitric  acid, 
no  coagulation  should  occur. 

If  it  be  desired  to  compare  the  powers  of  two  preparations, 
they  may  be  tested  side  by  side  as  above  described  and  portions 
of  each  withdrawn  and  acidulated  at  frequent  intervals.  The 
rapidity  with  which  the  end  point  is  reached  will  then  give  an 
indication  of  the  comparative  proteolytic  power. 

METT  METHOD 

This  method  consists  in  allowing  a  solution  of  the  proteolytic 
enzyme  to  act  upon  the  ends  of  a  column  of  coagulated  egg 
albumin  contained  in  a  narrow  glass  tube  and  observing  the 
rate  at  which  the  column  is  shortened  by  the  digesting  of  the 
coagulum.  While  this  cannot  be  considered  a  strictly  quanti- 
tative method,  it  has  obvious  advantages  as  a  means  of  demon- 
strating the  presence  or  absence  of  proteolytic  enzyme  or  any 
pronounced  difference  in  proteolytic  power  between  substances 
which  it  is  desired  to  compare. 

For  this  method  obtain  the  white  of  a  fresh  egg,  cut  it  thor- 
oughly with  scissors  or  stir  it  with  an  equal  volume  of  water, 
and  filter  or  strain  through  muslin  or  cheesecloth.  The  albumin 
thus  prepared  should  be  homogeneous,  nearly  clear,  and  entirely 
free  from  air  bubbles.  Fill  some  clean,  dry  capillary  glass 
tubes  of  1  to  2  mm.  diameter  with  the  prepared  albumin. 


326  METHODS    OF   ORGANIC   ANALYSIS 

This  may  be  done  either  by  drawing  up  the  fluid  into  the  tube 
as  into  a  pipette  or  by  lowering  the  tube  into  a  column  of  the 
fluid.  Great  care  must  be  taken  to  avoid  air  bubbles  in  filling 
the  tubes.  In  order  to  coagulate  uniformly  the  contents  of  a 
capillary  tube,  hold  it  (after  filling  with  albumin)  in  the  same 
manner  as  a  pipette  and  with  the  finger  over  the  upper  end 
touch  the  lower  end  to  the  surface  of  the  boiling  water  in  a 
water  bath  until  coagulation  begins,  then  lay  the  tube  hori- 
zontally in  the  boiling  water  ;  after  15  minutes  immersion  in 
boiling  water,  allow  the  tube  of  coagulated  albumin  to  cool 
slowly,  preferably  under  water,  then  remove  and  if  the  tube  is 
not  to  be  used  at  once,  seal  the  ends  by  means  of  sealing  wax  or 
paraffin.  For  use  cut  the  tube  of  coagulated  albumin  into 
sections  about  2  cm.  in  length  (being  careful  to  scratch  the 
tube  at  right  angles  to  its  axis  and  to  break  it  with  all  possible 
care  to  secure  clean-cut  square  ends).  Reject  the  end  sections 
and  any  which  contain  air  bubbles  or  in  which  the  albumin  has 
shrunken  and  does  not  completely  fill  the  capillary.  Immerse 
a  perfect  section  of  "  Mett  tube  "  thus  prepared  and  selected  in 
a  measured  volume  of  the  liquid  to  be  tested,  with  "  blanks  " 
alongside  in  which  all  of  the  conditions  are  the  same  except 
that  the  enzyme  is  omitted,  and  note  the  length  of  column 
digested  out  of  the  tube  after  standing  10  hours  at  38°  C.  or 
for  such  time  and  at  such  temperature  as  may  best  meet  the 
requirements  of  the  particular  case. 

When  the  test  is  applied  to  a  liquid  (e.g.  to  gastric  juice) 
an  equal  volume  of  the  same  liquid,  boiled  to  destroy  the 
enzyme,  may  be  used  for  the  blank  test. 

In  testing  a  solid,  it  should  be  dissolved  in  an  acid,  alkaline, 
or  neutral  medium  according  to  the  nature  of  the  enzyme  sup- 
posed to  be  present.  For  pepsin  or  trypsin  the  acidity  or  alka- 
linity prescribed  respectively  in  the  above  methods  from  the 
U.  S.  Pharmacopoeia  may  be  used,  and  comparisons  may  be 
made  with  a  sample  of  pepsin  or  trypsin  whose  power  is  known 
to  approximate  the  requirement  of  the  Pharmacopoeia  test. 

See  also  the  methods  of  expressing  proteolytic  power  given 
by  Rose  and  others  in  the  journal  articles  cited  below. 


PROTEINS   AND    PROTEASES  327 


ALLEN  METHOD  MODIFIED 

Dissolve  1  gram  of  dry  powdered  egg  albumin  in  20  cc.  of 
lukewarm  water  in  a  100-cc.  flask,  heat  in  a  boiling  water  bath 
to  coagulate  the  albumin  (Note  1),  and  cool  to  40°  C.,  add  0.1 
gram  of  the  sample  of  pepsin  to  be  tested  and  25  cc.  of  tenth- 
normal  hydrochloric  acid  (Note  2)  ;  warm  to  40°  C.,  and  main- 
tain at  this  temperature  for  3  hours  (Note  3) ;  then  add  a 
volume  of  tenth-normal  sodium  carbonate  solution  exactly 
equivalent  to  the  acid  previously  added  (Note  4),  mix  thor- 
oughly by  shaking,  and  place  the  flask  in  a  water  bath  at  90°  C. 
for  10  minutes  to  destroy  the  enzyme  and  coagulate  any  meta- 
protein  (syntonin)  which  may  have  been  dissolved  by  the  acid 
and  liberated  when  the  latter  was  neutralized  by  the  alkali. 
Cool,  dilute  with  water  to  100  cc.,  and  filter  through  a  dry 
paper,  protecting  the  solution  from  unnecessary  exposure  to  air 
so  that  it  shall  not  change  by  evaporation.  This  filtrate  (A) 
contains  the  proteoses,  peptones,  peptids  and  ammo-acids  which 
have  been  formed  by  digestive  hydrolysis  of  the  protein  plus 
what  existed  in  the  pepsin  added.  Measure  25  cc.  of  filtrate 
A  into  a  Kjeldahl  flask  and  determine  total  nitrogen.  To  50 
cc.  of  filtrate  A  (representing  one  half  of  the  solution),  add 
powdered  zinc  sulphate  in  the  cold  with  thorough  stirring  until 
the  solution  is  saturated,  allow  to  stand  with  occasional  stirring 
for  half  an  hour,  filter  through  pure  filter  paper,  and  wash  the 
precipitate  with  a  saturated  solution  of  zinc  sulphate.  Trans- 
fer the  precipitate  with  the  paper  to  a  Kjeldahl  flask  and  deter- 
mine nitrogen  as  a  measure  of  the  proteoses  ;  acidulate  the 
filtrate  (B)  with  hydrochloric  acid  and  precipitate  with  bro- 
mine as  described  above  under  determination  of  proteins  by 
bromine  precipitation.  The  nitrogen  of  the  bromine  precipi- 
tate serves  as  a  measure  of  the  amount  of  peptones  ;  the  filtrate 
from  the  bromine  precipitate  (filtrate  C)  contains  the  ammo- 
acids  and  any  peptids  not  precipitated  by  bromine.  The 
amount  of  nitrogen  in  these  forms  may  be  found  by  difference. 
From  the  total  nitrogen  of  proteoses,  peptones,  peptids,  and 
amino-acids  as  found  from  an  aliquot  portion  of  filtrate  A 


328  METHODS  OF  ORGANIC  ANALYSIS 

above,  subtract  the  nitrogen  of  proteoses  (zinc  sulphate  pre- 
cipitate) and  of  peptones  (bromine  precipitate)  ;  the  remainder 
is  the  nitrogen  of  peptids  and  amino-acids. 

A  blank*  test  should  be  conducted,  using  a  boiled  solution  of 
the  pepsin,  but  in  all  other  respects  exactly  as  above  described. 
Such  a  blank  test  will  supply  the  combined  corrections  for  the 
nitrogen  of  the  pepsin  and  for  the  cleavage  products  which  may 
possibly  be  produced  by  the  action  of  the  hydrochloric  acid  on 
the  coagulated  albumin. 

For  data  illustrating  the  application  of  this  method  see  Allen's 
Commercial  Organic  Analysis,  Volume  IV  (2d  ed.),  pp.  357- 
358. 

Note  1.  —  The  coagulated  albumin  should  be  in  the  form  of 
finely  divided  flocks  so  as  to  be  readily  accessible  to  the  enzyme. 
So  far  as  the  action  of  the  enzyme  on  the  albumin  is  concerned, 
it  would  seem  better  to  leave  the  latter  uncoagulated  ;  but  when 
this  is  done  there  may  be  difficulty  in  filtration  at  the  end  of 
the  digestion  period. 

Note  2. — The  addition  of  acid  at  this  point  is  intended 
to  give  the  reaction  most  favorable  for  the  pepsin  activity. 
If  the  enzyme  under  test  works  best  in  a  neutral  solution, 
the  acid  may  be  omitted  ;  or  in  the  case  of  trypsin,  sodium 
carbonate  may  be  added  at  this  point  to  give  the  favorable 
degree  of  alkalinity. 

Note  3.  —  While  three  hours  is  a  suitable  time  for  digestion 
in  the  case  of  commercial  pepsin  such  as  Allen  was  engaged  in 
testing,  it  is  obvious  that  a  longer  or  a  shorter  period  of  diges- 
tion maybe  adopted  when  working  with  substances  of  much 
lower  or  much  higher  proteolytic  power. 

Note  4.  —  Compare  Note  2.  If  no  acid  was  added  at  the  be- 
ginning of  the  digestion,  no  alkali  should  be  added  here.  If  al- 
kali was  added  at  the  beginning  of  the  digestion,  an  equivalent 
amount  of  acid  must  be  added  at  the  end.  The  object  here  is 
to  restore  the  neutrality  of  the  solution  so  that  the  subsequent 
heating  and  filtration  will  remove  any  dissolved  but  undigested 
protein  along  with  the  undissolved  coagulated  albumin. 


PKOTEINS   AND    PROTEASES  329 


OTHER  METHODS 

As  indicated  above,  many  other  methods,  some  of  them  very 
promising,  have  been  proposed  for  the  measurement  of  proteo- 
lytic  power.  In  research  work  especially,  the  various  methods 
available  should  be  carefully  studied  with  a  view  to  the  selection 
of  the  one  best  adapted  to  the  particular  problem  in  hand.  The 
references  given  at  the  end  of  this  chapter  will  put  the  reader 
in  touch  with  the  literature  of  the  subject. 

REFERENCES 


ABPERHALDEN:   Biochemisches  Handlexicon. 

:   Handbuch  der  Biochemischen  Arbeitsmethoden. 

:   Lehrbuch  der  Physiologische  Chemie. 

ALLEN  :  Commercial  Organic  Analysis. 

COHNHEIM  :   Chemie  der  Eiweisskorper. 

FISCHER  :   Untersuchungen  iiber  Aminosauren,  Polypeptide,  und  Proteine. 

FRANKEL  :   Descriptive  Biochemie. 

HAMMARSTEN  :   Physiological  Chemistry  (translated  by  Mandel). 

HAWK  :   Practical  Physiological  Chemistry. 

KONIG  :   Chemie  der  Menschliche  Nahrungs-  und  Genussmittel. 

MANN  :   Chemistry  of  the  Proteids. 

NEUBERG  :    Der  Harn  und  Korperflussigkeiten. 

OPPENHEIMER  :   Die  Fermente  und  ihre  Wirkung. 

:    Handbuch  der  Biochemie. 

OSBORNE  :   Proteins  of  the  Wheat  Kernel. 

:   The  Vegetable  Proteins. 

PLIMMER  :   The  Chemical  Constitution  of  the  Proteins. 
SCHAEFER  :   Text-book  of  Physiology. 
SHERMAN  :   Chemistry  of  Food  and  Nutrition. 

II 

On  Proteins 

(See  also  the  references  given  in  the  text) 

1880-86.  STUTZER:  Untersuchungen  ueber  die  quantitative  Bestimmung 
des  Proteinstickstoffes  und  die  Trennung  der  Proteinstoffe  von 
anderen  in  Pflanzen  vorkommenden  StickstofrVerbindungen. 
Journ.f.  Landwirthschaft,  28,  103;  29,  473;  34,  151. 


330  METHODS   OF    ORGANIC   ANALYSIS 

SCHULZE  und  BARBIERI  :  Zur  Bestimmung  des  Eiweisstoffe  und  der 
nichteiweissartigen  Stickstoffverbiudungen  in  der  Pflanzen. 
Landw.  Vers.  Stat.,  26,  218. 

1896.  TELLER:  The  Quantitative  Separation  of  Wheat  Proteids.     Bui.  42, 

Ark.  Agl.  Expt,  Sta.,  p.  81. 

1897.  ALLEN  and  SEARLE  :  Improved  Method  of  determining  Proteid  and 

Gelatinoid  Substances.     Analyst,  22,  258. 

1898.  BAUMANN   UND  BOMER  :   Ueber  die  Fallung   der  Albumosen  durch 

Zinksulfat.     Z.  Nahr.-Genussm.,  1, 106. 

1899.  MALLET  :  Analytical  Methods  for  distinguishing  between  the  Nitro- 

gen of  Proteids  and  that  of  the  Simpler  Amids  or  Amino-acids. 
Bui.  54,  Div.  Chem.,  U.  S.  Dept.  Agriculture ;  Chem.  News,  80, 
117,  168,  179. 

VIVIAN  :  A  Comparison  of  Reagents  for  Milk  Proteids.  16th  Ann. 
llpt.  Wis.  Agricl.  Expt.  Sta.,  p.  179.  . 

1900.  BARNSTEIN  :  Ueber  eine   Modifikation   des  von    Ritthausen  vorge- 

schlagenen  Verfahrens  der  Eiweissbestimmung.  Landw.  Vers. 
Stat.,  54,  327;  Z.  Nahr.-Genussm.,  1901,  4,  688. 

1894-1901.  SCHJERNING  :  [A  series  of  papers  on  the  quantitative  separation 
and  precipitation  of  proteins] .  Z.  anal.  Chem.,  33,  263 ;  34, 
135;  35,  285;  36,  643;  37,  73,  413;  39,  545,  633. 

1900.  TRAPS  and  BIZZELL:  Metlwds  of  determining  Protein  Nitrogen  in 

Vegetable  Matter.     J.  Am.  Chem.  Soc.,  22,  709. 

1901.  HART  :  Ueber  die  Quantitative  Bestimmung  der  Spaltungsprodukte 

von  Eiweisskorpern.     Z.  physiol.  Chem.,  33,  347. 

1903.  TEBB  :  The  Precipitation  of  Proteins  by  Alcohol  and  Certain  Other 

Reagents.     J.  Physiol.,  30,  25. 
VAN  SLYKE  and  HART  :  Methods  for  the  Estimation  of  the  Proteo- 

lytic  Compounds,  contained  in  Cheese  and  Milk.     Bui.  215,  New 

York  Agl.  Expt.  Sta. ;   Am.  Chem.  J.,  29,  150. 
BIGELOW  :  Meat  and  Meat  Products,  U.  S.  Dept.  Agriculture,  Bur. 

Chem.,  Bui.  65,  pp.  10,  17;  Bui.  13,  Part  10,  p.  1396;  Bui.  81, 

p.  104. 

1904.  GRINDLEY  :  A  Study  of  the  Nitrogenous  Constituents  of  Meats,  U.  S. 

Dept.  Agriculture,  Bur.  Chem.,  Bui.   81,  p.  110;  J.  Am.  Chem. 

Soc.,  26,  1086. 

HASLAM  :  Separation  of  Proteins.     J.  Physiol.,  32,  267. 
SNYDER  :  The  Determination  of  Gliadin  in  Wheat  Flour  by  means 

of  the  Polariscope.     J.  Am.  Chem.  Soc.,  26,  263. 

1905.  CHAMBERLAIN:  Determination  of  Gliadin  and  Glutenin  in  Flour. 

U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  81,  p.  118;  Circular 
20,  p.  14. 

GRINDLEY  and  EMMETT  :  The  Chemistry  of  Flesh.  J.  Am.  Chem. 
Soc.,  27,  658. 


PROTEINS   AND    PROTEASES  331 

1906.  BIGELOW  and  COOK  :  The  Separation  of  Proteoses  and  Peptones  from 

the  Simpler  Amino  Bodies.     J.  Am.  Chem.  Soc.,  28,  1485. 
CHAMBERLAIN  :   Investigations  on  Properties  of    Wheat   Proteins. 

.7.  Am.  Chem.  Soc.,  28,  1657. 
MATHEWSON  :  The  Optical  Rotation  of  Gliadin  in  Certain  Organic 

Solvents.    J.  Am.  Chem.  Soc.,  28,  1482. 

1907.  AGREE  :  A  Formaldehyde  Color  Test  for  Proteins.     Am.   Chem.  J., 

37,  604. 

COOK  and  TRESCOTT  :  A  Modification  of  the  Tannin-Salt  Method. 
J.  Am.  Chem.  Soc.,  29,  605. 

1908.  BARDACH  :  A  New  Protein  Reaction.     Z.  physiol.  Chem.,  54,  355. 
LIEBERMANN  :    (Formaldehyde   Color  Reaction  for  Protein).      Z. 

Nahr.-Genussm.,  16,  231. 

SEAMAN  and  GIES:  An  Examination  of  Bardach's  New  Protein  Test. 
Proc.  Soc.  Exp.  Biol.  Med.,  5,  125;  Chem.  Abs.,  2,  2829. 

1910.  OSBORNE:   Die    Pflanzenproteine.      Ergebnisse    der    Physiologic,   10, 

47-215. 

WEYL  :  (Precipitation  of  proteins  and  some  amino-acids  by  acetone). 
Z.  physiol.  Chem.,  65,  246.  Also  Ber.,  43,  508. 

1911.  MICRO  :  (Examination  of  Meat  Juices).     Z.  Nahr.-Genussm.,  20,  537. 
OSBORNE  and  GUEST:  (Analysis  of  the  Products  of  Hydrolysis  of 

Proteins).     /.  Biol.  Chem.,  9,  333,  425. 

VAN  SLYKE:  The  Analysis  of  Proteins  by  Determination  of  the 
Chemical  Groups  Characteristic  of  the  Diiferent  Amino-acids. 
/.  Biol.  Chem.,  10,  15. 

On  Proteases 

1881.  ROBERTS  :  Estimation  of  the  Amylolytic  and  Proteolytic  Activity  of 
Pancreatic  Extracts.  Proc.  Royal  Soc.,  32,  145. 

1897.  ALLEN:  (Valuation  of  Pepsin).  Pharm.  J.,  1897,  561;  Z.  anal. 
Chem.,  42,  466. 

1901.  KRUGER :  Quantitative   Observations   on   Pepsin -Action.     Z.   Biol., 

1901,  41,  467;  J.  Chem.  Soc.,  1902,  82,  ii,  33. 
SAMOJLOFF  :  Mett's  Method  of   estimating   Peptic  Activity.     Arch. 

ges.  Physiol.  (Pfluger},  85,  86;  J.  Chem.  Soc.,  1901,  80,  ii,  401. 
THOMAS  and  WEBER  :    Quantitative   Determination    of  Proteolytic 

Power.     Centrbl.    f.    Stoffwechselversuche  und    Verdauungskrank- 

heiten,  901,  2,  365;  Z.  Nahr.-Genussm.,  1902,  5,  723. 

1902.  PECKELHARING  :  On  Pepsin.     Z.  physiol.  Chem.,  35,  8. 

SPRIGGS  :  New  Method  of  Determining  Pepsin  Activity.  Z.  physiol. 
Chem.,  35,  465. 

1903.  WINOGRADOW:   Quantitative    Experiments  with  Peptic   Digestion. 

Z.  Nahr.-Genussm.,  6,  589. 


332  METHODS   OF   ORGANIC    ANALYSIS 

1905.  COBB  :  Contribution  to  our  Knowledge  of  the  Action  of  Pepsin,  with 

Special  Reference  to  its  Quantitative  Estimation.  Am.  J.  Physiol., 
13,  448. 

LOEHLEIN  :  Volhard's  Titrimetric  Method  for  Estimation  of  Pepsin 
and  Trypsin.  Beitr.  chem.  Physiol  Path.,  7,  120. 

1906.  LEVENE   and   ROUILLER  :     Estimation   of    Tryptophan   in    Protein 

Cleavage  Products.    J.  Biol.  Chem.,  2,  481. 

ROBERTSON  :  (Hydrolysis  of  Casein  by  Trypsin).  J.  Biol.  Chem.,  2t 
318. 

1907.  ABDERHALDEN  and  KOELKER  :  Employment  of  Optically  Active  Poly- 

peptids    as   Proof  of  the  Activity  of  Proteolytic  Enzymes.     Z. 
physiol.  Chem..,  51,  294. 

KUTTNER  :  The  Volhard  Method  for  Estimation  of  Pepsin.  Z. 
physiol.  Chem.,  52,  63. 

1908.  ARRHENIUS  :   Law  of  Schiitz  and  Reaction  Velocities.    Medd.  Vetens- 

kapsakademiems  Nobelinst.,  1,  No.  9 ;  Chem.  Abs.,  2,  2589. 
EINHORN:   Simplification  of  Jacoby-Solm  Ricin  Method  of  Pepsin 

Estimation.     Berlin,  klin.  Woch.,  45,  1567. 
FULD  and  LEVISON  :   Determination  of  Pepsin  by  Means  of  Edestin 

Test.     Biochem.  Z.,  6,  473. 
GOODMAN:    Ricin   Method  of  Jacoby-Solms  for  the   Quantitative 

Estimation  of  Pepsin.     Am.  J.  Med.  Sci.,  136,  734. 
GROSS  :   Activity  of  Trypsin  and  a  Simple  Method  for  its  Estimation. 

Arch.  exp.  Path.  Pharm.,  58,  157;  Chem.  Abs.,  2,  1570. 
MEYER:   Is  the  Schiitz  Law  for  Peptic  Digestion  Invalid?    Berlin. 

klin.  Woch.,  45,  1485. 
SORENSEN  :    (Estimation  of  Proteolytic  Power  by  Titration  of  Amino- 

acids).     Biochem.  Z.,  7,  45;   Chem.  Abs.,  2,  1288. 
WITTE  :    (Modification  of  Jacoby-Solms  Method).   Berlin,  klin.  Woch., 

1908,  p.  643. 

1909.  BERG  :   Comparative  Study  of  Digestibility  of  Different  Proteins  in 

Pepsin-Hydrochloric-Acid  Solutions.     Am.  J.  Physiol.,  23,  420. 
DEZANI  :   Contribution  to  the  Study  of  Pepsin.     Atti  della  R.  Accad. 

della  Scienze  di  Torino,  45,  225. 
LIEBERMANN:   New  Method  for  Clinical  Determination  of  Pepsin. 

Med.  Klin.,  1909,  1784. 

1910.  BLOOD  :   The  Erepsin  of  the  Cabbage   (Brassica  oleracea.)     J.  Biol. 

Chem.,  8,  215. 

FRANK  :  Digestibility  of  White  of  Egg  as  Influenced  by  the  Tempera- 
ture at  which  it  is  Coagulated.  J.  Biol.  Chem.,  9,  463. 

HAT  A  :  Estimation  of  Pepsin  by  the  Clearing  of  Turbid  Solutions  of 
Egg  Albumin.  Biochem.  Z.,  23,  179. 

KOELKER:  The  Study  of  Enzymes  by  Means  of  Synthetical  Poly- 
peptids.  J.  Biol.  Chem.,  8,  145. 


PROTEINS    AND    PROTEASES  333 

MENDEL  and  BLOOD  :    Some  Peculiarities  of  the  Proteolytic  Activity 

of  Papain.     J.  Biol.  Chem.,  8,  177. 
PALLADIN:    (Trypsin  Activity  and  its  Determination).     Arch.  ges. 

PhysioL  (Pftiiger),  134,  337;   Chem.  Abs.,  5,  1615. 
ROSE  :   Modified  Method  for  Clinical  Estimation  of  Pepsin.     Arch. 

Intern.  Med.,  5,  459;  Chem.  Abs.,  4,  1980. 
1911.   ABDERHALDEN  and  STEINBECK  :   Study  of  the  Action  of  Pepsin.     Z. 

physiol.  Chem.,  68,  293. 
AMBERG  and  JONES  :   On  the  Application  of  the  Optical  Method  to 

a  Study  of  the  Enzymatic  Decomposition  of  the  Nucleic  Acids. 

J.  Biol.  Chem.,  10,  81. 
GRABER  :   Some  Observations  upon  the  Assay  of  Digestive  Ferments. 

/.  Ind.  Eng.  Chem.,  3,  919. 
KOBER  :   A  Method  for  the  Study  of  Proteolytic  Ferments.     J.  Biol. 

Chem.,  10,  9. 
RAMSAY:   Method  of  Determining  the  Tryptic  Value  of  Pancreatin. 

J.  Ind.  Eng.  Chem.,  3,  822. 


CHAPTER   XVI 
Grain  Products 

IN  the  routine  analysis  of  vegetable  foods  and  feeding-stuffs 
it  is  customary  to  determine  moisture,  fat,  protein,  fiber,  and  ash 
and  to  estimate  the  remaining  substances  as  "  carbohydrates  by 
difference"  or  as  "nitrogen-free  extract."  Often  the  separate 
determination  of  fiber  is  omitted,  and  this,  as  well  as  the  sugars, 
starches,  pentosans,  etc.,  is  included  in  the  "carbohydrates  by 
difference." 

The  present  chapter  will  include  the  methods  for  such  deter- 
minations, which  are  of  fairly  general  application  in  food  anal- 
ysis, and  also  some  special  methods  for  the  examination  of  grain 
products  in  particular. 

PREPARATION  OF  SAMPLES 

Samples  for  analysis  should,  if  moist,  be  weighed  into  large 
flat-bottom  dishes,  dried  until  brittle  (though  not  necessarily 
quite  to  constant  weight)  at  a  temperature  not  above  that  of 
the  water  oven,  then  allowed  to  stand  exposed  to  the  air  until 
they  neither  gain  or  lose  in  weight,  and  the  weight  of  the  air-dry 
material  recorded  in  percentage  of  the  weight  of  the  fresh  sub- 
stance. The  air-dry  material  is  then  ground  in  a  coffee  or 
drug  mill l  until  all  will  pass  through  a  sieve  of  one-half-milli- 
meter mesh,  or,  if  this  is  not  feasible,  through  a  sieve  having 
round  holes  one  millimeter  in  diameter. 

Fineness  of  grinding  is  important,  not  only  to  secure  suffi- 
ciently accurate  portions  for  the  separate  determinations,  but 
also  to  permit  of  efficient  extraction  in  the  determinations 
described  below. 

1  In  laboratories  where  many  such  samples  are  to  be  ground  a  special  power 
mill  is  sometimes  provided. 

334 


GRAIN   PRODUCTS  335 


DETERMINATION  OF  MOISTURE  AND  FAT 


Dry  2  grams  for  5  hours,  or  to  constant  weight,  at  the  tem- 
perature of  boiling  water,  if  possible  in  a  current  of  dry  hydro- 
gen or  in  vacuo.  Consider  the  loss  of  weight  as  moisture. 
Extract  the  dried  sample  in  a  Soxhlet  or  continuous  extractor, 
with  anhydrous  alcohol-free  ether  for  sixteen  hours.  Dry  the 
extract  to  constant  weight  in  a  boiling  water  oven.  The  ether 
extraction  should  be  carried  out  at  a  distance  from  any  free 
flame,  the  flask  being  heated  by  a  safety  water  bath  or,  more 
conveniently,  by  an  electric  heater. 

Notes.  —  The  ether  extract  of  vegetable  substances  often 
contains  in  addition  to  fat  more  or  less  of  coloring  matters 
and  resinous  substances,  these  being  more  readily  soluble 
in  ether  containing  fatty  oils  than  in  ether  alone.  In  the 
cereal  grains  and  especially  in  the  milling  products  from 
which  the  outer  layers  of  the  grains  have  been  separated 
the  amount  of  such  impurities  is  usually  negligible.  If  the 
extract  is  made  to  percolate  a  layer  of  animal  charcoal,  prac- 
tically pure  fat  is  obtained.1 

The  reason  for  drying  in  a  current  of  hydrogen  rather  than 
in  air  is  that  the  oils  of  the  cereal  grains  belong  to  the  "semi- 
drying  "  group  and  therefore  absorb  oxygen  when  exposed  to 
air,  especially  at  high  temperature.  This  will  of  course  increase 
the  weight  of  the  fat  and  make  the  apparent  percentage  of 
moisture  too  low.  The  partially  oxidized  oils  are  also  apt 
to  be  incompletely  extracted  by  ether.  For  a  full  discus- 
sion of  the  determination  of  water  in  foods  and  physiological 
preparations,  see  Benedict  and  Manning:  Am.  J.  Physiol., 
1905,  13,  309. 

Fat  may  also  fail  of  complete  extraction,  even  when  un- 
changed, by  being  occluded  or  mechanically  inclosed  in  car- 
bohydrate or  protein  material  which  is  impervious  to  the 
ether. 

Although  ether  extracts  may  be  evaporated  at  the  tempera- 
ture of  boiling  water  without  loss  of  fat,  such  loss  has  been 

1  Patterson:  Am.  Chem.  J.,  12,  261. 


336  METHODS  OF  ORGANIC  ANALYSIS 

found  to  occur  in  drying  moist  samples  even  in  a  current  of 
hydrogen.  That  the  loss  in  such  cases  is,  in  part  at  least,  due 
to  an  actual  volatilization  of  material  may  be  shown  by  passing 
the  current  of  hydrogen  in  which  the  sample  is  dried  into  strong 
sulphuric  acid.  This  loss  is  probably  due  to  the  action  of  the 
escaping  steam  and  may  be  practically  avoided  by  drying  at  a 
lower  temperature,  preferable  in  a  partial  vacuum. 

All  three  of  the  causes  just  mentioned  tend  toward  a  defi- 
ciency of  fat  in  the  analysis  of  cooked  foods  prepared  from 
cereal  products.  Thus  in  a  number  of  experiments  on  bread 
making l  the  fat  found  by  analysis  of  the  dried  bread  was  less 
than  half  of  that  contained  in  the  materials  used,  and  the  iodine 
figure  of  the  ether  extract  of  the  bread  was  only  60.4  as  against 
101.4  in  that  of  the  original  flour,  showing  that  a  very  consider- 
able oxidation  had  taken  place  even  in  that  portion  of  the  fat 
which  was  still  soluble  in  ether. 

Berntrop's  method  for  the  determination  of  fat  in  breadstuffs 
is  as  follows :  2  Mix  150  grams  of  fresh  bread  with  500  cc.  of 
water,  add  100  cc.  of  concentrated  hydrochloric  acid,  and  boil 
for  two  hours  connected  with  a  reflux  condenser.3  Cool  the 
resulting  brown  liquid  to  room  temperature,  filter, with  suction 
through  a  moistened' fat-free  paper,  and  wash  with  cold  water. 
Dry  the  paper  and  residue  for  an  hour  at  100°  to  110°,  remove 
the  residue  as  completely  as  possible  from  the  filter  paper, 
and  grind  it  with  sand  in  a  mortar.  Cut  up  and  add  the 
filter  paper,  and  transfer  the  dry  mixture  to  a  paper  extrac- 
tion thimble  and  treat  with  ether  or  petroleum  ether  in  an 
extractor. 

Dormeyer's  method^  designed  originally  for  the  determi- 
nation of  fat  in  animal  tissues,  has  been  adapted  to  vege- 
table foods  by  Beger.5  From  3  to  5  grams  of  substance 
are  mixed  with  480  cc.  of  water,  20  cc.  of  25  per  cent  hy- 

1  Bui.  67,  Office  of  Experiment  Stations,  U.  S.  Dept.  Agriculture. 

2  Z.  angew.  Chem.,  1902,  121. 

8  In  treating  meal  or  flour,  heat  for  an  hour  in  a  water  bath  and  then  boil  for 
an  hour  with  the  reflux  condenser  attached. 

4  Arch.  ges.  Physiol.  (Pfluger),  1895,  61,  341  ;  1896,  65,  90. 

5  Chem.  Ztg.,  1902,  26,  112. 


GRAIN   PRODUCTS  337 

drochloric  acid,  and  1  gram  of  fat-free  pepsin.  The  mixture 
is  kept  at  37°  to  40°  for  twenty-four  hours,  filtered  with 
suction  through  a  paper  supported  on  a  porcelain  plate  and 
covered  with  asbestos,  and  both  the  filtrate  and  the  residue 
extracted  with  ether. 

DETERMINATION  OF  CRUDE  FIBER  1 

Extract  2  grams  of  the  substance  with  ordinary  ether,  or  use 
the  residue  from  the  determination  of  the  ether  extract.  To 
this  residue,  in  a  500-cc.  flask,  add  200  cc.  of  boiling  1.25  per 
cent  sulphuric  acid;  connect  the  flask  with  an  inverted  con- 
denser, the  tube  of  which  passes  only  a  short  distance  beyond 
the  rubber  stopper  into  the  flask.  Boil  at  once,  and  continue 
the  boiling  for  30  minutes.  A  blast  of  air  conducted  into  the 
flask  may  serve  to  reduce  the  frothing  of  the  liquid.  Filter, 
wash  with  boiling  water  till  the  washings  are  no  longer  acid; 
rinse  the  substance  back  into  the  same  flask  with  200  cc.  of  a 
boiling  1.25  per  cent  solution  of  sodium  hydroxide,  practically 
free  from  sodium  carbonate ;  boil  at  once,  and  continue  the 
boiling  for  30  minutes  in  the  same  manner  as  directed  above 
for  the  treatment  with  acid.  Filter  on  a  Gooch  crucible,  and 
wash  with  boiling  water  till  the  washings  are  neutral;  dry  at 
110°;  weigh;  incinerate  completely.  The  loss  of  weight  is 
crude  fiber. 

The  filter  used  for  the  first  filtration  may  be  linen,. one  of  the 
forms  of  glass  wool  or  asbestos  filters,  or  any  other  form  that 
secures  clear  and  reasonably  rapid  filtration.  The  solutions  of 
sulphuric  acid  and  sodium  hydroxide  are  to  be  made  up  of  the 
specified  strength,  determined  accurately  by  titration  and  not 
merely  from  specific  gravity. 

DETERMINATION  OF  ASH 

Char  about  2  grams  and  burn  to  whiteness  at  the  lowest  pos- 
sible red  heat,  preferably  in  a  flat-bottomed  platinum  dish  in  a 
muffle. 

1  Bui.  107,  Revised,  Bur.  Chem.,  U.  S.  Dept.  Agriculture.     See  also  Thatcher: 
J.  Am.  Chem.  Soc.,  1902,  24,  1210;  Browne:  Ibid.,  1904,25,  315. 
z 


338  METHODS  OF  ORGANIC  ANALYSIS 

If  considerable  quantities  of  phosphates  are  present,  these 
may  fuse  over  some  of  the  carbon  and  render  its  combus- 
tion very  slow.  In  such  cases,  extract  the  charred  mass 
with  a  little  hot  acetic  acid,  set  aside  the  solution  till  the 
char  is  burned,  then  evaporate  it  to  dry  ness  in  the  same 
dish  and  heat  the  residue  to  dull  redness  till  the  ash  is  white 
or  nearly  so.  Samples  containing  added  salt  should  be  ex- 
tracted with  water  before  charring,  and  the  determination 
finished  as  just  described. 

DETERMINATION  OF  PROTEIN 

Determine  total  nitrogen  by  one  of  the  modifications  of  the 
Kjeldahl  method  as  described  in  Chapter  XIV. 

On  the  assumption  that  proteins  in  general  contain  approxi- 
mately 16  per  cent  of  nitrogen,  it  has  been  customary  to  multi- 
ply the  percentage  of  nitrogen  by  6.25  as  an  estimate  of  the 
percentage  of  protein. 

The  extended  investigations  by  Osborne  and  his  associates,1 
and  by  Ritthausen,2  have  shown  that  nearly  all  the  cereal  pro- 
teins contain  over  16  per  cent  of  nitrogen,  so  that  the  results 
obtained  by  multiplying  the  nitrogen  by  6.25  are  too  high. 
The  factors  now  regarded  as  most  nearly  correct  are :  for 
wheat,  rye,  and  barley,  5.7  3 ;  for  maize,  oats,  rice,  and 
buckwheat,  6.00.  The  old  factor  6.25  is,  however,  still  fre- 
quently used  for  the  sake  of  uniformity  or  for  comparison 
with  earlier  work.  In  reporting  results,  therefore,  the  factor 
used  should  always  be  given. 

SEPARATION  OF   WHEAT   PROTEINS 

Nearly  all  the  nitrogenous  material  in  wheat,  and  especially 
in  wheat  flour,  is  in  the  form  of  proteins. 

The  proteins  of  wheat  have  been  studied  with  -great  thorough- 

1  Reports  Conn.  Agl.  Expt.  Station,  1890  et  seq.     Much  of  the  work  has  also 
appeared  in  J.  Am.  Chem.  Soc.,  Am.    Chem.  J.,  Am.  J.  Physiol.,  or  J.  Biol. 
Chem. 

2  Summarized  in  Landw.  Vers.  Stat.,  1896,  47,  391. 

3  The  factor  5.68  has  recently  been  proposed  for  wheat  flour. 


GRAIN    PRODUCTS  339 

ness  by  Osborne,  who  finds  five  forms  and  estimates  the  amount 
of  each  in  average  wheat  as  follows: 

f  Albumin  (leucosin)       0.3-0.4  per  cent 

Soluble  in  water  4  _  ^  ,      , . 

[Proteose  (about) 0.3  per  cent 

Soluble  in  ten  per  cent  NaCl  —  Globulin  (edestin)      .     .      0.6-0.7  per  cent 

Soluble  in  70  per  cent  alcohol — Gliadin  (  about ) 4.25  per  cent 

Insoluble  in  neutral  solvents — Glutenin 4.0-4.5  per  cent 

In  fine  flour  the  relative  amount  of  gliadin  is  higher  than  in 
the  whole  grain,  from  one  half  to  three  fifths  of  the  total  nitrogen 
of  fine  flour  being  usually  in  the  form  of  alcohol-soluble  protein. 

The  gliadin  and  glutenin  together  make  up  the  gluten,  which 
to  the  bread  maker  is  of  greater  importance  than  the  other  pro- 
teins of  the  flour.  In  addition  to  the  percentage  of  gluten  in  the 
flour  the  proportion  of  gliadin  in  the  gluten  is  important  to  its 
baking  qualities. 

Hence  the  protein  separations  of  direct  importance  in  establish- 
ing the  commercial  value  of  the  flour  are  (1)  to  separate  the 
water-  and  salt-soluble  proteins  from  the  gluten,  (2)  to  determine 
the  alcohol-soluble  protein  or  gliadin.  Knowing  the  amounts  of 
these  and  the  total  protein  present,  the  amount  of  glutenin  may 
be  found  by  difference. 

On  the  basis  of  Osborne's  studies,  Chamberlain  has  developed 
the  following  methods  for  the  routine  determination  of  alcohol- 
soluble  and  salt-soluble  proteins. 

Determination  of  alcohol-soluble  protein  {"crude  gliadin" ). — 
Treat  5  grams  of  the  sample  with  250  cc.  of  alcohol,  70  per  cent 
by  volume,  for  24  hours,  shaking  every  half  hour  during  the  first 
8  hours;  filter  through  a  dry  paper,  determine  nitrogen  in  100 
cc.  of  the  filtrate,  and  multiply  the  result  by  5.7. 

In  making  this  determination  of  nitrogen  the  100  cc.  of  solu- 
tion may  be  transferred  to  a  Kjeldahl  flask,  3  cc.  of  sulphuric 
acid  added,  and  the  liquid  boiled  down  to  a  small  volume,  after 
which  the  remainder  of  the  acid  is  added  and  the  determination 
completed  as  usual.  This  determination  will  usually  give  a 
result  very  slightly  higher  than  the  true  amount  of  gliadin,  since 
any  amino-acids  or  amids  present  are  likely  to  be  dissolved  by  the 
alcohol. 


340  METHODS   OF   ORGANIC    ANALYSIS 

Determination  of  salt-soluble  protein. — Treat  12  grams  of  the 
sample  with  300  cc.  of  5  per  cent  potassium  sulphate  as  described 
under  the  determination  of  alcohol-soluble  protein.  Determine 
nitrogen  in  100  cc.  of  the  filtrate  and  multiply  by  5.7  to  estimate 
the  salt-soluble  proteins. 

SEPARATION  OF  CARBOHYDRATES  IN  CEREAL  PRODUCTS 

Determination   of  Reducing   Sugars,  Sucrose,   Dextrin,   Starch, 
Pentosans,  and  Cellulose 

The  following  scheme  1  provides  for  each  of  the  substances  or 
groups  mentioned  and  avoids  the  danger  (inherent  in  any  plan 
of  making  a  number  of  independent  determinations)  of  includ- 
ing the  same  substance  as  a  constituent  of  more  than  one  group. 

Free  the  sample  from  fat  by  washing  with  ether. 

Extract  with  boiling  alcohol. 

Solution  A.  —  Evaporate  the  alcohol,  dilute  with  water,  and 
determine  the  reducing  power  of  portions  of  the  solution  before 
and  after  hydrolysis.  Calculate  the  reducing  sugar  and  sucrose. 

Residue  A.  —  Extract  with  cold  water. 

Solution  B.  —  Hydrolyze  a  portion  and  determine  the  result- 
ing dextrose,  calculate  dextrin  and  soluble  starch.  In  another 
portion,  precipitate  soluble  starch  by  barium  hydroxide,2  filter, 
and  determine  dextrin  in  the  filtrate. 

Residue  B.  —  Boil  with  water  and  treat  with  malt  extract  or 
saliva,  filter,  and  wash  thoroughly. 

Solution  O.  —  Hydrolyze,  determine  resulting  dextrose,  and 
calculate  starch. 

Residue  O.  —  Boil  with  2  per  cent  hydrochloric  or  sulphuric 
acid  until  the  maximum  reducing  power  of  the  solution  is 
reached.3 

Solution  D.  —  Determine  reducing  power  in  the  same  manner 
as  for  dextrose.  Calculate  as  xylose,  the  reducing  power  of 

1  Based  on  the  following  papers  :  Stone  :  J.  Am.  Chem.  Soc.,  1897,  19,  183. 
Sherman :  Ibid.,  1897,  19,  291.     Browne  and  Beistle  :  Ibid.,  1901,  23,  229. 

2  Asboth:   Chem.  Ztg.,  1889,  13,  591. 

8  For  sulphuric  acid  this  was  found  to  be  4  to  6  hours.  Stone  prefers  hydro- 
chloric acid  and  states  that  the  reaction  is  nearly  complete  in  1  hour. 


GRAIN   PRODUCTS  341 

which  is  1.03  times  that  of  dextrose.1  From  the  pentose  thus 
found  calculate  the  percentage  of  pentosan.  In  the  case  of 
wheat  it  has  been  found  2  that  the  material  ("  hemicellulose  ") 
dissolved  and  hydrolyzed  at  this  point  is  entirely  pentosan. 
The  same  is  probably  true  of  the  other  cereals.  The  pentosan 
thus  dissolved  and  hydrolyzed  does  not  include  necessarily  the 
entire  furfural-yielding  substance  of  the  cereal. 

Residue  D.  —  Boil  for  30  minutes  with  1  per  cent  sodium 
hydroxide,  filter  and  wash,  press  out  most  of  the  water,  and  ex- 
pose the  moist  fiber  to  chlorine  gas  for  one  hour.  Wash  with 
water,  boil  with  a  solution  containing  2  per  cent  sodium  sulphite 
and  0.2  per  cent  sodium  hydroxide;  filter,  wash  with  warm 
water  until  the  washings  are  neutral  and  colorless,  then  wash 
with  strong  alcohol,  dry,  and  weigh.  Deduct  the  ash  which  the 
residue  contains  and  calculate  the  organic  matter  as  cellulose. ,3 

Determination  of  Maltose,  Dextrin,  and  Starch  in  Malted  Cereal 

The  absence  of  any  considerable  amount  of  dextrose  or 
invert  sugar  must  be  shown  by  stirring  some  of  the  sample 
with  about  ten  times  its  weight  of  water  and  testing  the  fil- 
tered extract  by  means  of  phenylhydrazine  or  Barfoed's  solu- 
tion as  described  in  Chapter  III.  If  110  monosaccharide  is 
present,  the  percentages  of  maltose,  dextrin,  and  starch  can 
be  estimated  as  follows  : 

Mix  5  grams  of  sample  with  125  cc.  of  cold  water  4  in  a  250-cc. 
flask  ;  allow  to  stand  at  room  temperature  for  one  hour,  shaking 
frequently  ;  fill  to  the  mark,  shake,  and  filter  through  dry  paper. 
Determine  reducing  power  of  one  or  more  25-cc.  portions  of 

1  Stone  :  Am.  Chem.  J.,  1891,  13,  82.    Since  the  reducing  power  of  arabinose 
does  not  differ  greatly  from  that  of  xylose,  this  calculation  would  still  be -nearly 
correct  in  case  both  pentoses  were  present. 

2  J.  Am.  Chem.  Soc.,  1897,  19,  294. 

3  This  is  the  method  of  Cross  and  Bevan.   For  a  comparison  of  this  with  other 
methods  see  J.  Am.  Chem.  Soc.,  1897,  19,  304. 

4  If  the  sample  contains  an  active  enzyme,  some  of  the  carbohydrate  may  be 
changed  during  this  treatment  with  water.     To  prevent  this  a  very  dilute  alkali 
solution,  containing  0.02  per  cent  potassium  hydroxide  or  an  equivalent  amount 
of  sodium  or  ammonium  hydroxide,  may  be  used.     Ling  and  Rendle  ;  J.  Inst. 
Brewing,  1904,  10,  238  ;  Abs.  J.  Chem.  Soc.,  1904,  86,  ii,  507. 


342  METHODS   OF   ORGANIC    ANALYSIS 

this  filtrate  by  either  Defren's  or  Allihn's  method  and  calculate 
the  amount  of  maltose.1  Measure  50  cc.  of  the  same  filtrate  into 
a  100-cc.  flask,  add  5  cc.  of  hydrochloric  acid  of  1.125  sp.  gr.,  and 
hydrolyze  as  in  the  determination  of  starch.  Determine  the  re- 
sulting dextrose,  deduct  the  amount  due  to  maltose,  and  estimate 
the  remainder  as  due  to  dextrin.  Soluble  starch  if  present  would 
be  counted  as  dextrin  in  this  analysis. 

Treat  another  portion  of  the  original  sample  as  described  under 
the  determination  of  starch,  but  without  extracting  the  soluble 
carbohydrates.  From  the  dextrose  found,  subtract  that  yielded 
by  maltose  and  dextrin,  and  estimate  the  remainder  as  derived 
from  starch. 

The  results  require  a  slight  correction  on  account  of  the  pres- 
ence of  the  insoluble  residue  when  the  solution  is  diluted  to  vol- 
ume in  the  graduated  flask.  Although  some  details  of  the  method 
are  open  to  criticism,  it  gives  results  sufficiently  exact  for  the 
purpose  for  which  it  is  mainly  used,  which  is  to  show  whether 
the  starch  of  the  cereal  has  been  largely  changed  to  soluble 
products. 

The  same  plan  may  be  used  in  the  examination  of  cereal  foods 
prepared  by  parching  or  in  other  ways,  provided  only  one  reduc- 
ing sugar  is  present  in  appreciable  quantity.  The  amount  of 
soluble  carbohydrate  in  such  preparations  is  usually  too  small 
for  satisfactory  determination  by  means  of  the  polariscope. 

ACIDITY 

Acidity  in  flour  is  objectionable  both  as  an  indication  of 
deterioration  and  because  it  acts  upon  the  gliadin,  injuring  the 
physical  properties  which  are  especially  important  in  bread 
making. 

To  determine  acidity,  shake  10  grams  of  the  dry  sample 
with  100  cc.  of  cold  water,  filter,  and  titrate  an  aliquot  part 
with  tenth-normal  sodium  or  potassium  hydroxide,  using 
phenolphthalein  as  indicator.  In  fine  flour  the  acidity  calcu- 
lated as  lactic  acid  should  not  exceed  0.10  per  cent. 

1  If  Allihn's  method  is  used,  assume  the  reducing  power  of  maltose  to  be  0.61 
that  of  dextrose. 


GRAIN   PRODUCTS  343 


INTERPRETATION  OF  RESULTS 

Official  Definitions  and  Standards1 

Grain  is  the  fully  matured,  clean,  sound,  air-dry  seed  of 
wheat,  maize,  rice,  oats,  rye,  buckwheat,  barley,  sorghum, 
millet,  or  spelt. 

Meal  is  the  sound  product  made  by  grinding  grain. 

Flour  is  the  fine,  sound  product  made  by  bolting  wheat  meal 
and  contains  not  more  than  13. 5  per  cent  of  moisture,  not  less 
than  1.25  per  cent  of  nitrogen,  not  more  than  1.0  per  cent  of 
ash,  and  not  more  than  0.50  per  cent  of  fiber. 

Grraham  flour  is  unbolted  wheat  meal. 

Grluten  flour  is  the  product  made  from  flour  by  the  removal 
of  starch  and  contains  not  less  than  5.6  per  cent  of  nitrogen 
and  not  more  than  10  per  cent  of  moisture. 

Maize  meal,  corn  meal,  or  Indian  corn  meal  is  meal  made  from 
sound  maize  grain  and  contains  not  more  than  14  per  cent  of 
moisture,  not  less  than  1.12  per  cent  of  nitrogen,  and  not 
more  than  1.6  per  cent  of  ash. 

Rice  is  the  hulled  and  polished  grain  of  Oryza  sativa. 

Oatmeal  is  meal  made  from  hulled  oats  and  contains  not 
more  than  8  per  cent  of  moisture,  nor  more  than  1.5  per  cent 
of  crude  fiber,  not  less  than  2.24  per  cent  of  nitrogen,  and  not 
more  than  2.2  per  cent  of  ash. 

Rye  flour  is  the  fine  sound  product  made  by  bolting  ryemeal 
and  contains  not  more  than  13.5  per  cent  of  moisture,  not  less 
than  1.36  per  cent  of  nitrogen,  and  not  more  than  1.25  per  cent 
of  ash. 

Buckwheat  flour  is  bolted  buckwheat  meal  and  contains  not 
more  than  12  per  cent  of  moisture,  not  less  than  1.28  per  cent 
of  nitrogen,  and  not  more  than  1.75  per  cent  of  ash. 

Where  percentages  are  given  in  these  standards  they  are,  of 
course,  intended  to  represent  normal  limits  rather  than  averages 
or  extreme  limits. 

1  Circular  No.  19,  Office  of  the  Secretary,  U.  S.  Dept.  Agriculture. 


344 


METHODS  OF  ORGANIC  ANALYSIS 


Composition  of  Entire  Grains 

Wiley1  estimates  the    approximate    composition  of  average 
typical  American  grains  as  follows  : 

TABLE  25. —  PERCENTAGE  COMPOSITION  OF  ENTIRE  GRAINS  (WILEY) 


Barley 

Buck- 
wheat 

Maize 

Oats 

Rice 

unhulled 

Eye 

Wheat 

Moisture     

10.85 

12.00 

10.75 

10.00 

10.50 

10.50 

10.60 

Protein  (Nitrogen  x  6.25) 
Fat  (Ether  extract)     .     . 
Crude  fiber      

11.00 

2.25 
3.85 

10.75 

2.00 
10.75 

10.00 

4.25 
1.75 

12.00 
4.50 
12.00 

7.50 
1.60 
9.00 

12.25 
1.50 
2.10 

12.25 
1.75 
2.40 

Ash    

2.50 

1.75 

1.50 

3.50 

4.00 

1.90 

1.75 

Carbohydrates  (diff.)  .     . 

69.45 

62.75 

71.75 

58.00 

67.40 

71.75 

71.25 

Composition  of  Mitt  Products 

An  extended  study  of  the  mill  products  of  wheat,  made  by 
Teller  at  the  Arkansas  Experiment  Station,  1894  to  1898,2  in- 
cluded a  milling  experiment  in  which  the  principal  products  of 
a  long  process  (7  break)  roller  mill  were  analyzed  with  the 
following  results  : 

TABLE  26.  —  PERCENTAGE  COMPOSITION  OF  MILL  PRODUCTS  OF  WHEAT 

(TELLER) 


Patent 
Flour 

Straight 
Flour 

Low 

Grade 
Flour 

Ship 
Stuff 

Bran 

Whole 
Wheat 

Pure 
Germ 

Moisture 

13.75 

1390 

1322 

12  °5 

1285 

1390 

680 

Ash  . 

33 

47 

90 

3  12 

5  80 

2  15 

4.65 

Crude  fiber 

17 

26 

74 

3  55 

6  14 

2  17 

1.60 

Fat  .  .  . 

1  05 

125 

1  70 

4  80 

5  °0 

2  15 

1438 

Protein  (Nitrogen  x  5.7) 
Carbohydrates  (diff.)  .     . 

Total  Nitrogen  .... 
Protein  Nitrogen  .  .  . 
Amid  Nitrogen  .... 

9.69 
75.01 

1.70 
1.65 
.05 

10.37 
73.75 

1.82 
1.72 
.10 

12.88 
70.56 

2.26 
2.20 
.06 

16.36 
59.02 

2.87 
2.68 
.19 

15.56 
54.45 

2.73 
2.51 
.22 

12.31 
63.32 

2.16 
1.98 
.18 

36.00 
36.55 

6.34 

3 

!Bul.  45,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 

2Buls.  42  and  53,  Ark.  Expt.  Station  (Fayetteville,  Ark.). 

3  The  germ  is  richer  in  amid  nitrogen  than  other  parts  of  the  wheat  kernel. 


GRAIN    PRODUCTS 


345 


Under  the  system  of  milling  now  practiced  in  the  Northwest 
a  number  of  "  streams  "  of  flour  are  produced  which  are  after- 
ward united  in  different  proportions  to  form  the  market  grades 
of  flour.  Snyder  has  recently  published 1  the  following  analyses 
of  the  different  "  streams  "  as  obtained  in  milling  No.  1  North- 
ern wheat  by  typical  modern  machinery. 

TABLE  27.  —  SXYDER'S  ANALYSES  OF  DIFFERENT  MILL  PRODUCTS  FROM 
ONE  SAMPLE  OF  WHEAT 


Name  of  sample,  or  "  stream  " 

Water. 
Per  cent 

Ash. 
Per  cent 

Gliadin. 
Number  2 

Acidity. 
Per  cent 

Protein 
(N  x  6.25) 
Per  cent 

Carbo- 
hydrates 
and  fat. 
Per  cent 

90  per  cent  Patent  .     .     . 

10.89 

.48 

60.75 

.09 

13.38 

75.25 

Clear  grade    . 

10.53 

.85 

54.63 

.13 

14.19 

74  43 

First  break 

11.68 

.61 

60.83 

.09 

13.56 

i  tr.:rO 

74  1  i 

Second  break      .... 

11.10 

.52 

59.17 

.09 

15.00 

I   J  .  -I  *J 

73.38 

Third  break  .... 

10.97 

.49 

59.09 

.10 

16.50 

72.04 

Fourth  break      .... 

11.14 

.71 

58.31 

.11 

18.44 

69.71 

First  germ 

10.90 

.48  ' 

54.17  ' 

.08 

12.00 

76  69 

I  U.U*rf 

Second  germ  

10.37 

.59 

56.44 

.10 

12.63 

76.41 

First  middlings  .... 

10.37 

.42 

57.43 

.08 

12.63 

76.57 

Second  middlings  .     .     . 

10.69 

.42 

63.85 

.08 

13.31 

75.58 

Third  middlings     .     .     . 

10.29 

.37 

66.67 

.08 

12.56 

76.78 

Fourth  middlings  .     .     . 

11.08 

.38 

64.29 

.07 

12.25 

76.29 

Fifth  middlings      .     .     . 

10.21 

.42 

62.44 

.09 

12.81 

76.55 

Sixth  middlings      .     .     . 

10.15 

.37 

57.97 

.09 

12.94 

76.53 

Seventh  middlings  .     .     . 

10.30 

.47 

59.16 

.10 

13.31 

79.92 

First  tailings      .... 

9.01 

.77 

50.00 

.12 

13.50 

76.72 

Second  tailings  .... 

9.54 

.65 

57.94 

.10 

13.38 

76.43 

Second  tailings  cut      .     . 

9.32 

.83 

54.88 

.11 

13.44 

76.42 

Shorts  duster      .... 

9.36 

1.61 

39.51 

.18 

15.19 

73.84 

Shorts  middlings    .     .     . 

9.79 

4.03 



— 

17.47 



Wheat  

13.07 

1.82 

14.25 

It  will  be  seen  that  a  sample  of  wheat  containing  13.07  per 
cent  moisture  and  2.28  per  cent  nitrogen  gave  streams  of  flour 
containing  from  9.01  to  11.68  per  cent  moisture,  and  from 
1.92  to  2.95  per  cent  of  nitrogen.  The  "gliadin  number,"  or 

1  Bui.  85,  Minn.  Agl.  Expt.  Station,  St.  Anthony  Park,  Minn.,  1904. 

2  Alcohol-soluble  nitrogen  in  percentage  of  the  total  nitrogen. 


346  METHODS  OF  ORGANIC  ANALYSIS 

percentage  of  the  total  nitrogen  existing  in  the  form  of  alcohol- 
soluble  proteins,  varied  from  39.51  to  66.67.  It  is  interesting 
to  note  that  some  of  the  streams  of  flour  thus  obtained  from 
average  wheat  in  the  ordinary  milling  process  contain  con- 
siderably more  nitrogen  than  is  sometimes  found  in  so-called 
gluten  and  diabetic  flours  obtained  in  the  market. 

For  additional  analyses  and  results  of  experiments  upon  the 
digestibility  and  nutritive  value  of  cereal  products  see  Bui.  13, 
Part  9,  Bureau  of  Chemistry,  and  Buls.  28,  67,  85,  101,  126, 
and  143,  Office  of  Experiment  Stations,  U.  S.  Department  of 
Agriculture. 

REFERENCES 


ALLEN  :  Commercial  Organic  Analysis. 

ATWATER  and  BRYANT  :  The   Chemical   Composition  of  American  Food 

Materials.     Bui.   28,   Revised,   Office  of   Experiment  Stations,   U.   S. 

Dept.  Agriculture. 
JAGO  :  Science  and  Art  of  Breadmaking,  Chemistry  and  Analyses  of  Wheat. 

:  Technology  of  Breadmaking. 

KONIG  :  Chemie  der  Menschliche-Nahrungs-  und  Genussrnittel. 

LEACH  :  Food  Inspection  and  Analysis. 

MAURIZIO  :  Getreide,  Mehl  und  Brot. 

OSBORNE  :  Proteins  of  the  Wheat  Kernel. 

SNYDER  :  Studies  in  Bread  and  Breadmaking,  Buls.  67,  101,  126,  Office  of 

Experiment  Stations,  U.  S.  Dept.  Agriculture. 

VOGL  :  Die  wichtigsten  vegetabilischen  Nahrungs-  und  Genussmittel. 
WILEY  :  Foods  and  their  Adulteration. 
WINTON  :  The  Microscopy  of  Vegetable  Foods. 

II 

1894.  OSBORNE  and  VOORHEES:  Proteins  of  Wheat.     /.  Am.   CJiem.  Soc., 

16,  524. 

1895.  WILEY:  Analyses   of  Cereals  collected  at  the  World's   Columbian 

Exposition.     U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  45. 

1897.  OSBORNE  :  The  Amount  and  Properties  of  the  Proteins  of  the  Maize 

Kernel.     J.  Am.  Chem.  Soc.,  19,  525. 

SHERMAN:  The  Insoluble  Carbohydrates  of  Wheat.     J.  Am.  Chem. 
Soc.,  19,  291. 

1898.  WILEY  et  al.:  Cereals  and  Cereal  Products.     U.  S.  Dept.  Agiiculture, 

Bur.  Chem.,  Bui.  13,  Part  IX. 


GRAIN   PRODUCTS  347 

1899.  KRAEMER  :  An  Examination  of  Commercial  Flours.     J.  Am.  Chem. 

Soc.,  21,  650. 

1900.  GUESS:  The   Gluten  Constituents  of  Wheat  and   Flour   and   their 

Relation  to  Bread-making   Qualities.     J.  Am.  Chem.    Soc.,  22, 
263. 

1904.  SNYDER:     Wheat  and  Flour  Investigations.      Bui.  85,  Minn.  Agl. 

Expt.  Station. 

1905.  COCHRAN:  Estimation  of  Fat  in  Infants'  and  Invalids'  Foods.     J. 

Am.  Chem.  Soc.,  27,  906. 
HARPER  and  PETER  :  Protein  Content  of  the  Wheat  Kernel.     Bui. 

113,  Kentucky  Agricultural  Experiment  Station. 
HOPKINS,  SMITH,    and   EAST  :    Breeding   Corn.      Bui.    100,   Illinois 

Agricultural  Experiment  Station. 
SNYDER  :  Testing   Wheat  Flour  for  Commercial  Purposes.     J.  Am. 

Chem.  Soc.,  27,  1068. 

1906.  BREMER  :  (Grading  of  Flour  according  to  its  Catalase  Content).     Z. 

Nahr.-Genussm.,  11,  569. 
CHAMBERLAIN:  Investigation  on  the  Properties  of  Wheat  Proteins. 

/.  Am.  Chem.  Soc.,  28,  1657. 

NORTON:  Crude  Gluten.     /.  Am.  Chem.  Soc.,  28,  8. 
WINTON:    Diabetic   Foods.      Ann.   Kept.    Conn.   Expt.    Sta.,    1906, 

p.  153. 

1907.  ALWAY  and  GORTNER  :  The  Detection  of  Bleached  Flours.    J.  Am. 

Chem.  Soc.,  29,  1503. 
AVERY:  A  Contribution  to  the  Chemistry  of  Bleached   Flour.     J. 

Am.  Chem.  Soc.,  29,  571. 

HARCOURT  :  Breakfast  Foods ;  their  Chemical  Composition,  Digesti- 
bility, and  Cost.  /.  Soc.  Chem.  Ind.,  26,  240,  and  Ontario  Dept. 

Agriculture,  Bui.  162. 
SHAW  :  Polariscopic  Method  for  Determination  of  Gliadin.     J.  Am. 

Chem.  Soc.,  29,  1747. 
THATCHER:  A  Comparison   of  Various  Methods  of  estimating  the 

Baking  Qualities  of  Flour.     J.  Am.  Chem.  Soc.,  29,  910. 
WOOD  :  Test  for  Strength  of  Wheat  Flour.     Nature,  75,  391 ;  Chem. 

Abs.,  1,  1150. 

1908.  BAKER   and   HULTON:    Considerations    affecting    the    Strength    of 

Wheat  Flours.     J.  Soc.  Chem.  Ind.,  27,  368. 

MATHEWSON:  On  the  Analytical  Estimation  of  Gliadin.  /.  Am. 
Chem.  Soc.,  30,  74. 

1909.  HERTY:  Rapid  Determination  of   Oil  in   Cottonseed   Products.     J. 

Ind.  Eng.  Chem.,  1,  76. 

HOLDFLEISS  and  WTESSLING  :  Laboratory  Experiments  on  the  De- 
termination of  the  Milling  and  Breadmaking  Qualities  of  Wheat. 
J.  Soc.  Chem.  Ind.,  28,  808. 


348  METHODS   OF   ORGANIC    ANALYSIS 

1910.  POLENSKE  :  The  Determination  of  Fat  in  Foods.     Arb.  kais.  Gesund- 

heitsamte,  33,  563;  Chem.  Abs.,  5,  325. 
WILLARD  :  Changes  in  Weight  of  Stored  Flour.     Kansas  State  Board 

of  Health,  7,  9;  Chem.  Abs.,  5,  1636. 
WINTON  :  Diabetic  Foods.     British   Food  «/.,  12,  23 ;  Chem.  Abs.,  4, 

1632. 

1911.  GREAVES  :  Some  Factors  influencing  the  Quantitative  Determination 

of  Gliadin.     /.  Biol.  Chem.,  9,  271. 

HOAGLAND  :  The  Determination  of  Gliadin  or  Alcohol-soluble  Pro- 
tein in  Wheat  Flour.     J.-Ind.  Eng.  Chem.,  3,  838. 


CHAPTER  XVII 


Milk 

Cows'  milk  is  concisely  described  as  essentially  an  aqueous 
solution  of  milk  sugar,  albumin,  and  certain  salts,  holding  in 
suspension  globules  of  fat  and  in  a  state  of  semisolution  casein 
together  with  mineral  matter  (Richmond).  Small  amounts  of 
other  compounds  are  also  present,  but  need  not  be  considered 
here. 

Standard  milk  (whole  milk)  is  defined1  as  the  lacteal  secre- 
tion obtained  by  the  complete  milking  of  one  or  more  healthy 
cows  properly  kept  and  fed,  excluding  that  obtained  within 
fifteen  days  before  and  five  days  after  calving,  and  contains  not 
less  than  8.5  per  cent  of  solids  not  fat,  and  not  less  than  3.25 
per  cent  of  milk  fat. 

These  limits  are  considerably  below  the  average  and  consider- 
ably above  the  lowest  authentic  figures  which  have  been  found. 
Average  milk  may  be  assumed  to  contain  12.9  to  13  per  cent 
of  total  solids,  made  up  of  — 


Fat 

Protein 

Milk  Sugar  2 

Ash 

In  round  numbers  3  
Estimated  average    

4.0 
4.0 

3.3 
3.35 

5.0 

4.88 

0.7 
0.72 

The  protein  content  of  average  milk  is,  therefore,  about  one- 
fourth  of  the  total  solids.  In  general  the  same  relation  holds 
in  milk  which  is  richer  than  the  average.  Each  increase  of  1 

1  Circular  No.  19,  Office  of  the  Secretary,  U.  S.  Dept.  Agriculture. 

2  The  figures  for  milk  sugar  include  the  small  amount  of  undetermined  non- 
nitrogenous  matter. 

3  These  are  the  figures  used  in  most  publications  of  the  U.  S.  Dept.  Agri- 
culture. 

349 


350 


METHODS    OF   ORGANIC    ANALYSIS 


per  cent  in  total  solids  thus  involves  on  the  average  an  increase 
of  0.25  per  cent  of  proteins,  the  remaining  0.75  per  cent  being 
practically  all  fat.  This  increase  in  proteins  and  fat  is  usually 
accompanied  by  a  slight  increase  in  ash  and  decrease  in  milk 
sugar.  The  following  average  percentages  illustrate  these 
relations  in  rich  milk : 


Total  Solids 

Fat 

Protein 

Milk 

Ash 

Sugar 

Average  for  5  years  ;    mixed  even- 

ing milk  of  400  to  500  cows    .     . 

14.62 

5.39 

3.66 

4.82 

0.75 

Average     of     13     unusually    rich 

samples  from  individual  cows 

18.03 

7.76 

4.68 

4.76 

0.83 

The  composition  of  milk  of  less  than  average  richness  cannot 
be  so  definitely  stated.  In  some  cases  there  is  a  deficiency  of 
fat  and  protein  with  no  decrease  in  milk  sugar,  while  in  other 
cases  the  reverse  is  true.  Usually  if  an  unadulterated  milk  is 
poor  in  fat,  it  will  be  found  proportionately  poor  in  protein; 
while  if  the  fat  is  normal,  the  protein  is  usually  also  normal  and 
the  low  percentage  of  solids  -not  fat  is  due  to  a  deficiency  in 
milk  sugar. 

Several  hundred  American  analyses,  made  before  1890,  com- 
piled and  averaged  in  ten  groups  arranged  according  to  per- 
centage of  total  solids,  gave  the  following  results  : 1 

TABLE  28.  —  COOKE'S  COMPILATION  OF  AMERICAN  ANALYSIS  OF  MILK 


Group 
No. 

Total 
solids. 
Per  cent 

Fat. 
Per  cent 

Proteins. 
Per  cent 

Sugar 
and  ash. 
Per  cent 

Group 
No. 

Total 
solids. 
Per  cent 

Fat. 
Per  cent 

Proteins. 
Per  cent 

Sugar 
and  ash. 
Per  cent 

1 

11.35 

3.20 

2.99 

5.16 

6 

13.71 

4.46 

3.48 

5.77 

2 

11.77 

3.36 

3.03 

5.38 

7 

14.25 

4.87 

3.65 

5.73 

3 

12.21 

3.60 

3.10 

5.51 

8 

14.77 

5.20 

3.87 

5.70 

4 

12.75 

3.82 

3.29 

5.64 

9 

15.17 

5.47 

4.07 

5.63 

5 

13.17 

4.09 

3.40 

5.68 

10 

15.83 

5.88 

4.26 

5.69 

1  Cooke  :  Vermont  Agricultural  Experiment  Station  Report  for  1890,  p.  97. 


MILK 


351 


As  a  rule  the  percentage  of  milk  sugar  and  ash  is  most  nearly 
constant,  that  of  fat  is  most  variable,  while  the  protein  varies 
with  the  fat,  but  to  a  much  smaller  extent.  The  variations 
which  may  be  regarded  as  usual,  and  the  extreme  variations 
Avhich  the  writer  has  found  authentically  recorded,  are  as  fol- 
lows : 


Fat. 
Per  cent 

Solids  not  fat. 
Per  cent 

Proteins. 
Per  cent 

Milk  sugar. 
Per  cent 

Ash. 
Per  cent 

Usual  variations    .     . 

3-6 

8.5-9.5 

3-4 

4.6-5 

0.7-0.78 

Extreme  variations  J  . 

1.04-14.67 

4.90-13.76 

2.86-9.98 

2.33-5.28 

0.66-1.44 

The  extreme  variations  are  of  no  practical  value  as  a  means 
of  determining  the  limits  within  which  milk  shall  be  considered 
unadulterated,  partly  because  it  is  possible  to  practice  "adulter- 
ation through  the  cow"  (i.e.  by  selection,  feeding,  and  manner 
of  milking  to  obtain  "  genuine  "  milk  much  below  the  normal 
quality),  but  mainly  because  the  milk  which  reaches  market  is 
practically  always  the  mixed  product  of  several  cows  so  that 
individual  variations  have  comparatively  little  effect. 

There  are  many  causes  of  variation  in  the  composition  of 
cows'  milk.  Only  the  most  important  can  be  given  here. 
Other  conditions  being  normal,  the  percentages  of  fat  and 
proteins  are  higher  in  autumn  and  winter  than  in  spring  and 
summer;  they  also  increase  as  the  amount  of  milk  decreases 
toward  the  end  of  each  period  of  lactation.  Milk  drawn  in  the 
evening  is  generally  0.3  to  0.4  per  cent  richer  in  fat  than  that 
obtained  in  the  morning,  and  at  any  one  milking  the  last 
portions  drawn  are  much  richer  than  the  first.  The  influence 
of  a  change  of  food  upon  the  percentage  composition  of  milk  is 
usually  only  temporary.  In  general  the  peculiarities  of  breed  2 

1  Including  only  results  obtained  from  apparently  healthy  cows,  believed  to 
have  been  milked  regularly  under  normal  conditions. 

2  For  comparison  of  the  milk  of  different  breeds  see  Richmond's  Dairy 
Chemistry,  pp.  122-126,  Report  of  the  New  York  State  Expt.  Station  for  1891 
(abstracted  in  the  Expt.  Station  Record,  4,  263) ,  and  Report  of  the  Wisconsin 
Expt.  Station  for  1901,  p.  85. 


352  METHODS    OF   ORGANIC    ANALYSIS 

and  the  qualities  of  individual  animals  are  the  most  important 
factors  in  determining  the  richness  -of  milk.  Aside  from  all 
these  conditions  the  milk  of  individual  cows  is  subject  to  con- 
siderable fluctuation,  especially  in  fat  content.  Thus  the 
analyses  of  60  monthly  samples  of  the  mixed  milk  of  about 
500  cows  showed  a  variation  of  0.89  in  the  percentage  of  fat, 
the  greatest  deviation  from  the  average  being  0.50  per  cent. 
About  one  half  of  the  determinations  of  fat  in  the  milk  of  in- 
dividual cows  of  the  herd  during  the  same  period  were  more 
than  0.50  per  cent  and  about  one  fifth  were  more  than  1.0  per 
cent  above  or  below  the  average.  Milk  representing  the  mixed 
product  of  several  farms,  such  as  is  now  commonly  sold  in  large 
cities,  should,  therefore,  be  much  more  uniform  in  composition 
than  that  of  a  single  cow  or  a  small  herd.1 

SAMPLING  AND  PRESERVATION  OF  SAMPLES 

If  the  lot  of  milk  to  be  sampled  is  small  it  can  be  mixed  by 
pouring  from  one  vessel  to  another  from  two  to  ten  times, 
according  to  the  extent  to  which  the  cream  has  separated,  and 
the  portion  for  analysis  dipped,  or  withdrawn  by  means  of  a 
pipette,  from  near  the  center.  When  the  sample  is  too  large  to 
be  handled  in  this  way  it  should  be  transferred  if  necessary  to 
a  cylindrical  can  and  sampled  by  means  of  a  Scovell  tube.2  In 
order  to  obtain  a  proper  sample  of  a  large  lot  of  milk  delivered 
in  cans  of  the  same  diameter,  it  is  only  necessary  to  sample  each 
can  with  the  Scovell  tube  and  mix  the  portions  thus  obtained. 

The  sample  should  be  placed  at  once  in  a  clean,  dry,  sterile 
bottle,  tightly  stoppered,  and  analyzed  as  soon  as  possible. 
Before  withdrawing  each  portion  for  analysis  the  sample  must 
be  thoroughly  mixed  by  pouring  —  not  by  shaking. 

1  Fuller  discussions  of  the  variations  in  the  milk  of  individual  cows  and  mixed 
milk  of  herds  will  be  found  in  some  of  the  reference  books  given  at  the  end  of 
the  chapter,  in  the  papers  on  the  composition  of  milk  published  annually  by 
Richmond  in  the  Analyst,  and  in  Hittcher's  Gesammtbericht  liber  die  Unter- 
suchung  der  Milch,  Berlin,  1899. 

2  Wiley's  Agricultural  Analysis,  Vol.  III.,  p.  470;  Leach's  Food  Inspection 
and  Analysis,  2d  ed.,  p.  131.     These  sampling  tubes  are  sold  by  dealers  in 
dairy  apparatus. 


MILK 


353 


If  the  analysis  cannot  be  made  at  once  or  if  the  sample  is  to 
be  kept  for  some  time  after  the  analysis,  it  must  either  be 
stored  at  a  temperature  near  the  freezing  point  or  preserved 
by  the  addition  of  an  antiseptic.  Formaldehyde 1  added,  while 
the  milk  is  still  fresh,  in  the  proportion  of  1 : 
1000  will  preserve  the  sample  for  months  with- 
out apparent  change.  This  amount  of  form- 
aldehyde has  a  scarcely  perceptible  influence 
upon  the  analytical  results.  If  preservation  for 
only  a  few  days  is  required,  a  smaller  amount 
of  formaldehyde  should  be  used,  1:  2000  to  1: 
10,000,  according  to  the  freshness  of  the  milk. 


\ 


PRELIMINARY    OR  PARTIAL    EXAMINATION 


DETERMINATION  OF  SPECIFIC  GRAVITY 

The  specific  gravity  of  milk  is  usually  be- 
tween 1.029  and  1.034.  Since  cream  is  con- 
siderably lighter  than  milk,  the  specific  gravity 
would  be  lowered  by  the  addition  of  water  or 
of  cream,  but  cases  in  which  genuine  milk 
shows  a  low  specific  gravity  as  a  result  of  high 
fat  content  are  very  rare.  As  already  ex- 
plained, high  percentages  of  fat  are  normally 
accompanied  by  high  percentages  of  proteins,  so 
that  in  most  cases  the  specific  gravity  is  higher 
in  rich  than  in  poor  milk.  With  practice  the 
samples  which  are  exceptions  to  this  rule  can 
usually  be  detected  by  noticing  the  apparent  FIG.  is.— Lactometer 

.,  .,        £  ,,          .„        '  ,  of      Quevenne     or 

viscosity  and  opacity  of  the  milk  as  it  runs  from  Soxhiet  type,  with 
the  surface  of  the  lower  bulb  of  the  lactometer.  thermometer  in  the 

stem. 
The  specific  gravity  taken  in  connection  with 

this  appearance  is  much  used  as  a  preliminary  test  by  milk  in- 
spectors and  is  recommended  by  Richmond  as  the  best  means 
of  rapidly  testing  each  lot  of  milk  received  by  a  large  dairy. 

1  Other  preservatives  are  sometimes  useful.     See  Richmond's  Dairy  Chem- 
istry, p.  144;  and  Grelat:   Chem.  Abs.,  1,  1588. 

2A 


354  METHODS   OF   ORGANIC    ANALYSIS 

The  Quevenne,  Veith,  and  Soxhlet  lactometers  are  hydrom- 
eters of  sufficient  range  for  use  with  milk  and  so  graduated 
as  to  read  the  "excess  gravity  "  over  water  taken  as  1000.  Thus 
a  milk  of  1.0315  specific  gravity  gives  a  lactometer  reading  of 
31.5°.  These  instruments  are  often  made  to  include  a  Fahren- 
heit thermometer,  the  scale  of  the  latter  being  on  the  same 
stem  with  the  lactometer  scale  (Fig.  18).  The  lactometer  read- 
ing should  be  taken  between  50°  and  65°  F.  and  corrected  for 
temperature  by  adding  or  subtracting  0.1°  for  each  degree  F. 
above  or  below  60.  The  New  York  Board  of  Health  lactometer 
has  a  scale  reading  zero  in  pure  water  and  100  at  1.029  specific 
gravity. 

VOLUMETRIC  DETERMINATION  OF  FAT 

Babcock,  in  1890,  introduced  the  first  satisfactory  rapid 
method  for  the  determination  of  fat  in  milk.  On  mixing  milk 
with  approximately  an  equal  volume  of  strong  sulphuric  acid, 
the  casein  is  dissolved  while  the  fat  remains  unchanged  and 
can  be  separated  by  centrifugal  force.  The  test  is  performed 
in  a  bottle  with  a  neck  so  graduated  that  the  percentage  of  fat 
can  be  read  off  directly  upon  removing  the  bottle  from  the 
centrifuge. 

Determination. — Measure  17.6  cc.  of  milk  at  14°  to  18° 
(about  55°  to  65°  F.)  and  introduce  into  the  test  bottle.  Add 
17.5  cc.  of  sulphuric  acid  of  1.82  sp.  gr.  (commercial  concen- 
trated acid  is  usually  the  right  strength),  allowing  the  acid  to 
flow  down  the  side  of  the  bottle  so  as  not  to  mix  with  the  milk. 
When  acid  has  been  added  to  all  of  the  bottles  and  everything 
is  ready  to  start  the  whirling,  mix  the  milk  and  acid  quickly 
and  thoroughly  by  shaking,  and  continue  the  shaking  until  the 
curd  is  in  solution  and  the  liquid  has  reached  a  permanent  and 
uniform  color.  Then  place  an  even  number  of  the  bottles  in 
opposite  pockets  in  the  machine  and  whirl  at  the  rate  of  800  to 
1200  revolutions  per  minute,  according  to  the  diameter  of  the 
wheel  which  carries  the  bottle.  After  whirling  five  minutes, 
fill  with  hot  water  to  the  shoulder  of  the  bottle  and  whirl  two 
minutes,  then  fill  with  hot  water  to  near  the  top  of  the  gradua- 
tion in  the  neck  and  whirl  again  for  two  minutes.  The  per- 


MILK  355 

centage  of  fat  is  now  shown  by  the  height  of  the  column  in 
the  graduated  neck  of  the  bottle. 

Notes.  —  The  capacity  of  the  graduated  neck  of  the  bottle 
from  0  to  10  is  2  cc.  It  is  assumed  that  the  17.6  cc.  of  milk 
taken  for  the  determination  will  weigh  10  times  as  much  as 
2  cc.  of  warm  butter  fat.  It  is  important  that  the  final  read- 
ings be  taken  while  the  fat  is  still  warm.  On  account  of  the 
unavoidable  contraction  of  the  fat  while  taking  these  readings 
it  is  customary  to  read  from  the  bottom  of  the  lower  to  the  top 
of  the  upper  meniscus.  The  result  is  usually  within  0.2  per 
cent  of  that  found  by  the  gravimetric  method.  The  column  of 
fat  should  be  of  a  clear  yellow  color  throughout.  If  the  acid 
used  is  too  weak,  flocks  of  undissolved  casein  are  apt  to  be 
found  in  the  lower  part  of  the  fat  column ;  if  too  strong,  the 
acid  may  char  the  fat.  For  a  full  discussion  of  the  details  of 
the  test,  with  directions  for  applying  it  to  other  dairy  products, 
see  the  work  of  Farrington  and  Woll.1 

The  most  important  modifications  of  the  Babcock  method  are 
fully  described  in  Richmond's  Dairy  Chemistry,  pp.  1.74-192. 

CALCULATION  OP  SOLIDS  FROM  SPECIFIC  GRAVITY 
AND  FAT 

Many  formulae  have  been  proposed  by  which  to  calculate  the 
percentage  of  solids  in  milk  from  the  percentage  of  fat  and  the 
specific  gravity.  The  results  thus  obtained  are  sufficiently  ac- 
curate for  many  technical  purposes  and  often  for  routine  in- 
spection work  which  is  not  to  be  made  the  basis  of  legal  action. 
Such  formulae  may  be  found  in  many  of  the  works  referred  to 
at  the  end  of  this  chapter.  They  are  necessarily  based  on  the 
assumption  that  each  per  cent  of  fat  causes  a  definite  decrease, 
and  each  per  cent  of  solids  not  fat  a  definite  increase,  in  the 
specific  gravity. 

Since  the  solution  densities  of  proteins,  milk  sugar,  and 
milk  ash  differ  considerably,2  any  change  in  the  relative 

1  Testing  Milk  and  Its  Products.     Madison,  Wisconsin. 

2  Allen  :  Commercial  Organic  Analysis,  Vol.  IV.  (2d  ed.),  p.  166. 


356  METHODS   OF   ORGANIC    ANALYSIS 

proportions  of  these  constituents  must  alter  the  solution 
density  of  the  solids  not  fat  and  thus  diminish  the  accuracy 
of  this  method. 

One  of  the  simplest  of  these  formulae  is  that  of  Richmond  : 
Total  solids  =  Lactometer  reading      ^  fet      Q  u> 

4 

With  samples  which  do  not  differ  greatly  from  the  average 
composition  the  results  thus  calculated  are  usually  accurate 
within  0.25  per  cent. 

DETERMINATION  OF  FAT,   PROTEINS,  MILK  SUGAR, 
AND  ASH 

TOTAL  SOLIDS  AND  ASH 

Into  an  accurately  weighed  flat-bottomed  platinum  dish  intro- 
duce two  to  five  grams  (depending  upon  the  size  of  the  dish  ;  see 
below)  of  the  thoroughly  mixed  milk  and  weigh  quickly  to  the 
nearest  milligram.  If  this  weighing  cannot  be  accomplished 
within  one  minute,  the  dish  should  be  covered  with  a  weighed 
watch  glass  to  retard  evaporation.  Place  the  open  dish  on  a 
water  bath  or  on  top  of  the  boiling  water  oven  until  nearly  all 
of  the  water  is  expelled  ;  dry  to  constant  weight  in  a  boiling 
water  oven  or  an  air  bath  kept  constantly  at  97°  to  100°.  The 
residue  is  somewhat  hydroscopic  and  must  be  weighed  quickly 
upon  removal  from  the  desiccator  in  order  to  obtain  the  correct 
amount  of  total  solids. 

Ignite  the  dry  solids  in  a  muffle  at  550°  to  600°,  or,  if  this  is 
not  feasible,  regulate  a  Bunsen  burner  to  give  a  very  small  col- 
orless flame  and  apply  this  carefully  with  constant  attention  so 
that  no  part  of  the  dish  is  heated  abcTve  the  lowest  possible  red- 
ness. The  ash  should  be  white  or  very  light  gray.  After 
obtaining  the  weight  of  the  ash  it  may  be  used  in  testing  for 
preservatives,  as  described  below. 

Notes  on  Total  Solids.  —  When  the  same  portion  is  not  to  be 
used  for  the  determination  of  ash  a  platinum  dish  is  not  essential. 
Lead  foil  bottle  caps  are  then  very  convenient,  as  they  are  eas- 
ily numbered  by  scratching,  quickly  heated  and  cooled,  and  so 


MILK  357 

cheap  that  each  dish  can  be  rejected  after  being  used  once.  In 
order  that  a  larger  surface  may  be  exposed,  the  dish  may  contain 
dry  sand  or  other  porous  material  and  a  small  stirring  rod 
weighed  with  the  dish  and  used  to  stir  the  residue  while  drying. 
Unless  absorbed  upon  porous  material,  no  more  than  0.5  gram  of 
milk  for  each  square  centimeter  of  the  area  of  the  bottom  of 
the  dish  should  be  taken  for  the  determination. 

The  methods  used  in  the  Government  Laboratory,  London, 
for  the  determination  of  solids  in  sour  or  fermented  milk  and 
the  estimation  of  the  solids  lost  in  fermentation  have  recently 
been  described  by  Thorpe.1 

Notes  on  Ash.  —  Normally  about  two-thirds  of  the  ash  of 
cows'  milk  is  insoluble  in  hot  water.  The  presence  of  a 
larger  proportion  of  soluble  ash  may  be  due  to  the  use  of 
mineral  preservatives  or  to  the  addition  of  salts  to  restore 
the  density  and  ash  content  to  milk  which  has  been  watered. 
Special  tests  for  some  of  the  mineral  preservatives  are  given 
beyond.  In  important  cases  it  may  be  necessary  to  analyze 
the  ash  to  show  whether  it  is  of  normal  character.  Richmond  2 
gives  the  following  as  the  average  composition  of  milk  ash  : 
calcium  oxide,  20.27  per  cent;  magnesium  oxide,  2.80  per 
cent;  potassium  oxide,  28.71  per  cent;  sodium  oxide,  6.67  per 
cent;  phosphoric  anhydride,  29.33  per  cent  ;  chlorine,  14.00 
per  cent ;  carbonic  anhydride,  0.97  per  cent  ;  sulphuric  an- 
hydride, trace  ;  ferric  oxide,  etc.,  0.40  per  cent.  According 
to  most  other  writers3  milk  ash  contains  sulphates,  but  only 
traces  of  carbonates.  A  sample  of  ash  from  the  mixed  milk  of 
about  500  cows  examined  by  Thompson  and  the  writer  showed 
no  appreciable  amount  of  carbonates  and  only  traces  of  sul- 
phates. When  the  ash  was  prepared  at  known  temperatures, 
there  was  no  volatilization  of  chlorides  below  650°,  but  even 
at  450°  to  500°  there  was  considerable  loss  of  chlorine,  due 
doubtless  to  the  formation  of  acid  products  in  the  combus- 
tion of  the  organic  constituents  of  the  milk. 

1J.  Chem.  Soc.,  1905,  87,  206. 

2  Dairy  Chemistry,  p.  32. 

8  See  tabulated  analyses  in  Stohmann's  Milch-  und  Molkereiproducte,  p.  89. 


358  METHODS  OF  ORGANIC  ANALYSIS 

FAT —  GRAVIMETRIC  DETERMINATION 

Adams'  Paper   Coil  Method 

In  this  method  the  milk  is  dried  on  porous  paper,  the  fat  ex- 
tracted by  means  of  ether  into  a  weighed  flask,  the  ether  evapo- 
rated, and  the  fat  weighed. 

Apparatus.  —  (1)  Strips  of  thick  absorbent  fat-free  paper  about 
55  cm.  long  and  6.25  cm.  wide,1  each  rolled  into  a  loose  coil 
and  fastened  by  means  of  a  piece  of  wire  or  fat-free  thread.  If 
difficulty  is  found  in  making  a  loose  coil,  two  pieces  of  fat-free 
string  may  be  laid  lengthwise  upon  the  paper  strip  before  rolling 
it  up.  This,  however,  should  not  be  necessary. 

(2)  A  Soxhlet  apparatus  for  ether  extraction,  the  form  hav- 
ing ground  glass  connections  being  recommended. 

(3)  A  safety  water   bath  or,  preferably,  an  electric  heater 
which  can  be  easily  regulated. 

Determination. — Mix  the  milk  thoroughly  and  absorb  a  known 
amount,  about  5  grams,  on  the  paper  coil.  The  milk  can  be  meas- 
ured by  means  of  a  5-cc.  pipette  and  delivered  directly  upon 
the  coil,  but  as  milk  is  more  viscous  than  water,  an  ordinary  5- 
cc.  pipette  will  deliver  less  than  5  cc.  of  milk,  so  that  this  method 
can  be  made  accurate  only  by  determining  experimentally  the 
amount  of  milk  which  the  pipette  actually  delivers.  A  better 
method  is  to  pour  about  5  cc.  into  a  very  small  beaker,  weigh 
quickly  to  centigrams  and  at  once  absorb  the  milk  by  standing 
the  coil  in  the  beaker.  The  absorption  can  be  hastened  by  in- 
clining the  beaker  and  rotating  the  coil.  The  last  drops  in  the 
beaker  must  be  carefully  absorbed.  Stand  the  coil  upon  the  dry 
end  and  reweigh  the  beaker  quickly  to  centigrams.  If  carried 
out  rapidly,  this  method  is  considerably  more  accurate  than 
measuring  with  a  pipette.  Dry  the  coil  thoroughly  in  a  boiling 
water  oven,  place  in  a  Soxhlet  extractor,  and  extract  with  an- 
hydrous ether,  using  the  electric  heater  or  safety  water  bath  and 
keeping  the  apparatus  as  far  as  possible  from  free  flames.  If 

1  Strips  of  paper  especially  prepared  for  this  purpose  are  made  by  Schleicher 
and  Schlill.  If  these  are  not  available,  the  paper  strips  must  be  very  carefully 
extracted  before  use.  See  Richmond's  Dairy  Chemistry,  pp.  91-93. 


MILK  359 

not  more  than  5  grams  of  milk  is  used  and  the  extractor  siphons 
at  intervals  of  10  to  15  minutes,  the  extraction  need  not  be  con- 
tinued longer  than  three  hours.  At  the  end  of  extraction  dis- 
connect the  apparatus,  remove  the  coil,  replace  the  extractor, 
recover  nearly  all  the  ether  by  allowing  it  to  collect  in  the  space 
formerly  occupied  by  the  coil,  return  the  ether  to  its  bottle,  and 
heat  the  flask  containing  the  fat  in  a  boiling  water  oven  until 
the  weight  is  practically  constant. 

Notes.  —  As  the  milk  is  absorbed  by  the  paper  the  greater 
part  of  the  fat  is  left  on  or  near  the  surface,  so  that  it  is  very 
rapidly  extracted  by  the  ether.  The  coil  must  be  thoroughly 
dried  before  extracting  with  ether.  The  drying  can  be  hastened 
by  pressing  in  the  dry  end  of  coil  so  that  the  inner  layers  of  the 
wet  end  are  made  to  project  in  the  form  of  a  cone.  Such  a  coil 
will  usually  be  dry  after  standing  two  to  three  hours  in  the  boil- 
ing-water oven.  On  removing  from  the  oven  press  back  the 
projecting  end  of  the  coil  and  place  it,  milk  end  down,  in  the 
extractor ;  connect  with  the  flask,  pour  in  ether  until  it  siphons 
into  the  flask,  then  enough  more  ether  to  cover  about  half  the 
coil.  This  is  sufficient  to  avoid  any  danger  of  the  flask  going 
dry  during  the  extraction,  if  the  heat  is  so  regulated  that  no 
perceptible  amount  of  ether  escapes  the  reflux  condenser.  To 
dry  the  extract,  leave  it  in  the  boiling  water  oven  for  three  hours, 
allow  to  cool  for  one  half  hour,  weigh,  and  then  repeat,  heating 
about  one  hour  each  time,  until  two  successive  weighings  show 
a  loss  of  less  than  one  milligram.  In  laboratories  where  many 
determinations  are  made  it  is  customary  to  dry  the  extract  for  a 
fixed  length  of  time  (usually  five  hours),  which  has  been  found 
by  experience  to  be  sufficient. 

Babcock  Asbestos  Method  l 

Provide  a  hollow  cylinder  of  perforated  sheet  metal,  60  mm. 
long  and  20  mm.  in  diameter,  closed  5  mm.  from  one  end  by  a 
disk  of  the  same  material.  The  perforations  should  be  about 
0.7  mm.  in  diameter  and  about  0.7  mm.  apart.  Fill  loosely 
with  from  1.5  to  2.5  grams  of  freshly  ignited,  woolly  asbestos, 
1  Bui.  107,  Bur.  Chem.,  U.  S.  Dept.  Agriculture. 


360  METHODS    OF   ORGANIC    ANALYSIS 

free  from  fine  and  brittle  material,  cool  in  a  desiccator,  and  weigh. 
Introduce  a  weighed  quantity  of  milk  (between  3  and  5  grams) 
and  dry  at  100°  to  constant  weight.  This  weight  shows  the 
percentage  of  total  solids.  Place  the  cylinder  in  an  extractor 
and  complete  the  determination  of  fat  as  described  above. 

This  method  avoids  the  possibility  of  having  any  ether- 
soluble  matter  in  the  porous  substance  used  to  absorb  the  milk. 
It  is  especially  recommended  for  the  determination  of  fat  in 
cream  which  cannot  be  absorbed  upon  the  paper  coil  without 
previous  dilution. 

PROTEINS 

Formerly  milk  proteins  were  precipitated,  or  the  milk  evapo- 
rated to  dryness,  and  the  residue  after  washing  with  ether  and 
dilute  alcohol  was  dried,  weighed,  burned,  and  the  ash  deducted. 
On  account  of  the  difficulty  of  completely  removing  the  sugar 
and  fat,  the  results  thus  obtained  were  usually  too  high ;  so 
that  in  the  older  statements  of  the  composition  of  milk  (some 
of  which  are  still  often  quoted)  the  proteins  were  usually  over- 
estimated. 

Protein  in  milk  is  now  calculated  from  the  nitrogen  content, 
multiplying  the  latter  by  the  usual  factor  6.25  or  sometimes  by 
a  special  factor,  6.33  or  6.3T,  based  on  analyses  of  milk  proteins 
showing  less  than  16  per  cent  of  nitrogen. 

To  determine  the  total  nitrogen  in  milk  pour  5  to  10  grams 
of  the  sample  into  a  small  beaker,  weigh  quickly  to  centigrams, 
pour  the  rnilk  carefully  into  a  Kjeldahl  flask,  re  weigh  the 
beaker,  and  introduce  20  to  25  cc.  of  concentrated  sulphuric 
acid  into  the  flask  in  such  a  way  as  to  wash  down  any  milk 
which  may  have  remained  in  the  neck.  Add  0.7  gram  of  mer- 
cury, heat  gently  over  a  very  small  flame  until  most  of  the 
water  is  expelled  and  no  more  frothing  or  spirting  occurs,  then 
increase  the  size  of  the  flame  and  complete  the  determination  as 
described  in  Chapter  XIV. 

Casein  can  be  precipitated  by  acidulating  the  milk  or  by 
means  of  magnesium  sulphate.  Determination  of  nitrogen  in 
the  washed  precipitate  shows  the  amount  of  casein  in  the  milk. 
Albumin  can  be  precipitated  by  boiling  the  filtrate,  and  deter- 


MILK  361 

mined  in  the  same  manner.  Detailed  directions  for  these 
determinations  will  be  found  in  Bulletin  107,  Bureau  of  Chem- 
istry, U.  S.  Department  of  Agriculture. 

MILK  SUGAR  OR  LACTOSE 

In  most  cases  the  direct  determination  of  lactose  is  unneces- 
sary, as  the  difference  between  the  percentage  of  total  solids  and 
the  sum  of  the  percentages  of  fat,  proteins,  and  ash  should  not 
differ  from  the  true  percentage  of  lactose  by  more  than  0.1  to 
0.2  per  cent.  When  direct  determination  is  desired,  either  the 
polariscopic  method  or  one  of  the  methods  based  upon  the 
reduction  of  copper  can  be  used.  In  the  former  case  the  pro- 
teins are  precipitated  and  the  solution  clarified  by  means  of 
mercuric  nitrate  or  iodide;  in  the  latter,  by  cupric  hydroxide  or 
acetic  acid,  alum,  and  aluminium  hydroxide. 

Optical  Determination 1 

Place  65.8  grams  of  milk  in  each  of  two  flasks,  one  graduated 
at  100,  the  other  at  200  cc.,  to  each  add  4  cc.  of  mercuric  nitrate 
solution,2  fill  to  the  mark,  shake,  filter  through  dry  paper,  and 
polarize  in  a  200-mm.  tube  in  the  Schmidt  and  Haensch  polar- 
iscope. 

In  each  case  the  reading  is  too  high  on  account  of  the  volume 
occupied  by  the  precipitate  which  contains  the  proteins  and  fat 
of  the  milk.  This  volume  is  twice  as  great  in  proportion  in  the 
100-cc.  as  in  the  200-cc.  flask.  The  corrected  reading  and  the 
volume  occupied  by  the  precipitate  can,  therefore,  be  calculated 
by  the  method  of  double  dilution  as  in  the  following  example  :  3 

Weight  of  milk  taken,  65.8  grams,  or  twice  the  "lactose 
normal "  weight 4  for  the  Ventzke  scale. 

1  Wiley  and  Ewell :  J.  Am.  Chem.  Soc.,  1896,  18,  428. 

2  To  prepare  this  solution  dissolve  mercury  in  twice  its  weight  of  nitric  acid, 
1.42  specific  gravity,  and  add  to  the  solution  an  equal  volume  of  water ;  or  pre- 
pare a  solution  of  equal  strength  by  dissolving  solid  mercuric  nitrate  in  water 
acidulated  with  nitric  acid. 

3  Compare  Wiley's  Agricultural  Analysis,  Vol.  Ill,  pp.  102,  278. 

4  Calculated  from  the  sucrose  normal  weight  and  the  approximate  specific 
rotatory  powers  of  sucrose  and  lactose.     These   data  and  directions  for  the 
manipulation  of  the  polariscope  have  been  given  in  Chapters  III  and  IV. 


362  METHODS    OF    ORGANIC    ANALYSIS 

Average  reading  from  100-cc.  flask,  10.45. 
Average  reading  from  200-cc.  flask,  5.075. 
Then 

10.45- (5.075x2)  =  0.30  (half  the  error  in  the  higher  reading). 
10.45-(0.30  x2)  =  9.85  (corrected  reading  for  100-cc.  flask). 

9.85  -j-  2  =  4.925,  corrected  percentage  of  lactose. 
The  volume  of  the  precipitate  is  calculated  as  follows : 
10.45 -r-  2  =  5.225,  apparent  percentage  of  lactose  (100-cc.  flask). 
Then  5. 225:  4. 925::  100:  a;. 

x  =  94.26,  the  volume  of  solution  in  the  100-cc.  flask.  Hence 
the  volume  of  the  precipitate  is  5.74  cc. 

Determination  by  Fehling  Solution* 

Dilute  25  cc.  of  milk  with  400  cc.  of  water  in  a  500-cc.  flask, 
add  10  cc.  of  the  copper  sulphate  solution  used  in  the  Fehling 
method,  mix  and  add  4.4  cc.  of  normal  sodium  or  potassium 
hydroxide  (or  an  equivalent  amount  of  a  weaker  standard  solu- 
tion), fill  to  the  mark,  mix,  and  filter  through  dry  paper.  The 
filtrate  must  contain  copper  in  order  to  insure  the  absence  of 
any  trace  of  free  alkali.  In  this  clear  filtrate  lactose  can  be 
determined  by  means  of  Fehling  solution  either  by  Defren's 
method  as  described  in  Chapter  III.,  or  by  Soxhlet's  method, 
Bui.  107,  I.  c.  The  milk  is  so  greatly  diluted  in  clarifying  the 
solution  that  the  volume  of  the  precipitated  proteins  and  fat 
can  be  neglected. 

If  lactose  is  to  be  determined  volumetrically,  the  proteins 
can  be  precipitated  and  the  solution  clarified  as  described  in 
Richards  and  Woodman's  Air,  Water,  and  Food. 

INTERPRETATION  OF  RESULTS 

The  principal  adulterations  affecting  the  percentages  of 
nutrients  in  milk  are  the  addition  of  water  (sometimes  con- 
taining dissolved  solids)  and  the  removal  of  cream.  These 

1  Bui.  107,  loc.  cit. 


MILK  363 

adulterations  are  sometimes  difficult  to  detect  with  certainty 
because  genuine  cows'  milk  varies  considerably  both,  in  fat  and 
in  other  solids.  Since  the  percentage  of  fat  is  more  variable 
than  that  of  solids  not  fat,  skimming  is  more  difficult  to  detect 
than  watering.  If  as  much  as  one  fourth  of  the  fat  were 
removed,  the  skimming  would  usually  be  indicated  by  the  dis- 
turbance of  the  normal  relation  between  the  percentage  of  fat 
and  that  of  proteins  or  of  solids  not  fat ;  but  the  analysis  can- 
not be  said  to  prove  the  removal  of  cream  unless  it  shows  a 
lower  percentage  of  fat  than  is  ever  found  in  genuine  normal 
milk.  Starting  with  average  milk  containing  4  per  cent  fat 
and  9  per  cent  solids  not  fat,  one  tenth  of  the  fat  could  be 
removed  by  skimming  and  the  resulting  product  containing 
3.6  per  cent  fat  could  not  be  distinguished  by  analysis  from 
genuine  milk ;  while  if  the  fat  were  reduced  to  3.6  per  cent  by 
watering,  the  solids  not  fat  would  be  reduced  to  8.1  per  cent, 
which  is  sufficiently  below  the  normal  to  be  detected  without 
difficulty.  Occasionally  genuine  milk  contains  even  less  than 
8.0  per  cent  of  solids  not  fat  (the  deficiency  in  most  of  these 
cases  falling  mainly  upon  the  milk  sugar),  so  that  the  limit  of 
8.5  for  solids  not  fat  might  indicate  watering  where  none  had 
been  practiced.  Such  errors  are  avoided  by  taking  account  of 
the  proteins  and  ash.  Milk  should  contain  not  less  than  8.5 
per  cent  of  solids  not  fat,  3.0  per  cent  of  proteins,  0.7  per 
cent  of  total  ash,  0.5  per  cent  of  ash  insoluble  in  hot  water. 
These  four  determinations,  especially  if  supplemented  by 
the  refractometer  examination  of  the  serum  as  described 
below,  will  usually  suffice  to  show  whether  the  milk  is  gen- 
uine or  has  been  watered  with  or  without  the  addition  of 
soluble  solids. 

In  most  cases  it  is  not  necessary  to  show  conclusively 
whether  milk  has  been  skimmed  or  watered,  but  only  whether 
it  meets  the  requirements  of  a  legal  or  trade  standard.  The 
principal  standards  in  force  in  the  United  States  in  1910  are 
given  in  the  accompanying  table,  from  the  Twenty-seventh 
Annual  Report,  Bureau  of  Animal  Industry,  U.  S.  Department 
of  Agriculture. 


364  METHODS   OF   ORGANIC   ANALYSIS 

TABLE  29.  —  UNITED   STATES  AND   STATE   STANDARDS  FOR   MILK,  1910 


State. 

Total  solids.  , 
Per  cent 

1  Solids  not  fat. 
Per  cent 

Fat.  Per  cent 

State. 

Total  solids. 
Per  cent 

Solids  not  fat. 
Per  cent 

1 
& 

i 

United  States  l  .    .     . 



8.5 

3.25 

New  Hampshire  .    . 

13 

9.5 

3.5 

California     .... 

— 

8.5 

3 

New  Jersey      .     .     . 

11.5 

— 

3 

Colorado  



8.5 

3.25 

New  Mexico 



Connecticut  .... 

11.75 

8.5 

3.25 

New  York   .... 

11.5 

— 

3 

Dist.  Columbia  .    .     . 

— 

9 

3.5 

North  Carolina    .     . 

— 

8.5 

3.25 

Delaware2    .... 

— 

— 

— 

North  Dakota      .     . 

12 

— 

3 

Florida  2  .     .     . 

Ohio         

12 



3 

12 

8.5 

3  25 

12  5 

3 

Hawaii      

11.5 

3.50 

Oregon              .     . 

12.2 

9 

3  2 

Idaho    

11 

8 

3 

Pennsylvania  2     .     . 

Illinois                .     .     . 

8.5 

3 

Porto  Rico 

12 



3 

8  5 

3  25 

Iowa 

12.5 

3 

South  Dakota 

13 

8.5 

3  25 

Kentucky      .... 

12 

8.5 

3.25 

Tennessee    .... 

8.5 

3.25 

Louisiana      .... 

13 

95 

3.5 

Texas  

8.5 

3.25 

11.75 

8.5 

3.25 

Utah                 •    •     • 

12 

9 

32 

Maryland      .... 

12.5 

3.5 

Vermont      .... 

12.5 

9.25 

4 

Massachusetts  .     .     . 

12.15 

— 

3.35 

May  and  June  .    . 

12 

— 

— 

Michigan  . 

12  5 

3 

8.5 

3  °5 

Minnesota     .... 

13 

3.5 

Washington     .     .     . 

12 

8.75 

3.25 

Missouri   .... 

8  5 

3  25 

8  5 

3 

Montana  

12 

9 

3 

"Wyoming 

12 

2  4 

Nebraska      .... 

3 

May  and  June  .     . 

11.5 

— 

1  Standards  of  Purity  of  Food  Products. 
Office  of  the  Secretary,  Circular  19. 
2  Municipal  control  ;  no  State  standard. 


United  States  Dept.  Agriculture, 


EXAMINATION  OF  MILK  SERUM  FOR  ADDED  WATER 
In  general  the  serum  or  whey  obtained  from  milk  under 
fixed  conditions  is  believed  to  be  much  more  uniform  in  prop- 
erties than  the  milk  itself,  in  which  case  watering  will  be  more 
certainly  detected  by  an  examination  of  the  whey  than  of  the 
whole  milk. 

Woodman's  method,  adopted  by  Leach,  for  preparing  the 
serum  is  as  follows  :  To  100  cc.  of  milk  at  room  temperature 
in  a  beaker,  add  2  cc.  of  25  per  cent  acetic  acid,  cover  and  heat 
in  a  water  bath  at  70°  C.  for  20  minutes  ;  then  place  the  beaker 


MILK  365 

in  ice  water  for  10  minutes,  after  which  filter  through  a  dry 
paper. 

This  should  result  in  a  clear  filtrate  (the  serum  or  whey) 
which  may  be  tested  either  for  specific  gravity  or  with  the  im- 
mersion refractometer.  A  specific  gravity  below  1.027  at  15° 
C.,  or  a  reading  of  the  refractometer  below  39  at  20°  C.,  is  a 
strong  indication  that  the  sample  is  watered. 

The  great  advantage  in  rapidity  and  convenience  of  such  a 
method  over  the  determination  of  solids  not  fat  commends  it 
to  inspection  laboratories  where  many  samples  must  be  rapidly 
examined  for  adulteration,  and  in  several  such  laboratories  the 
refractometer  reading  of  the  serum  is  now  taken  as  the  chief 
criterion  of  watering.  It  should  be  noted,  however,  that  the 
usual  legal  criterion  is  a  minimum  percentage  of  solids  not  fat. 

According  to  data  determined  by  Tice1  and  by  Leach2  it 
would  appear  that  milk  very  poor  in  solids  but  free  from  added 
water  may  fall  far  below  the  usual  legal  minimum  of  8.5  per 
cent  solids  not  fat  while  yielding  serum  readings  of  39  to  42 
on  the  immersion  refractometer  scale.  Hence  if  milk  is  de- 
clared watered  only  when  both  the  percentage  of  solids  not 
fat  and  the  refractometer  reading  of  the  serum  are  below 
minimum  limits,  there  will  be  much  less  danger  of  prosecutions 
for  watering  in  cases  of  milk  not  watered  but  naturally  poor  in 
solids. 

CHEMICAL   PRESERVATIVES 

The  chemical  preservatives  most  likely  to  be  used  in  milk  are 
formaldehyde,  hydrogen  peroxide,  boric  acid  or  borax,  and  fluo- 
rides/ Benzoates  and  salicylates  may  perhaps  be  used  in  rare 
instances.  Methods  for  the  detection  and  determination  of  these 
and  other  preservatives  will  be  found  in  the  next  chapter. 

Carbonate  or  bicarbonate  is  sometimes  added  to  milk,  not  as 
a  preservative  properly  so  called,  but  as  an  adulterant  to  hide 
the  fact  that  the  milk  has  undergone  acid  fermentation,  and 
so  to  give  it  a  fraudulent  appearance  of  freshness. 

When   milk   contains   the   equivalent   of    0.05    per  cent   of 

1  Report  of  the  New  Jersey  State  Board  of  Health,  1909,  pp.  191-194. 

2  Leach  :  Food  Inspection  and  Analysis,  2d  ed.,  pp.  166-169. 


366          METHODS  OF  ORGANIC  ANALYSIS 

sodium  carbonate,  the  ash  obtained  by  direct  ignition  of  the 
solids  shows  effervescence  on  addition  of  hydrochloric  acid. 
Such  effervescence  is  rarely  if  ever  seen  in  the  ash  of  pure 
milk,  but  since  Richmond  has  found  small  amounts  of  carbonic 
acid  in  the  ash  of  milk  believed  to  have  been  pure,  the  presence 
of  carbonate  or  bicarbonate  should  be  confirmed  by  applying 
Schmidt's  test,  in  which  10  cc.  of  milk  are  mixed  with  an  equal 
volume  of  alcohol  and  a  few  drops  of  a  1  per  cent  solution  of 
rosolic  acid.  The  color  is  brownish  yellow  in  pure  milk  but 
rose-red  in  milk  containing  carbonate  or  bicarbonate.  A  com- 
parative test  with  pure  milk  should  always  be  made.  The  re- 
action is  nearly  as  delicate  as  the  test  for  effervescence  in  the 

ash. 

REFERENCES 


ALLEN  :   Commercial  Organic  Analysis. 

CHAPIN:   Theory  and  Practice  of  Infant  Feeding. 

CONN  :   Bacteria  in  Milk  and  its  Products. 

FARRINGTON  and  WOLL  :    Testing  Milk  and  its  Products. 

FLEISCHMANN  :    Lehrbuch  der  Milchwirthschaft. 

GROTENFELT  :   The  Principles  of  Modern  Dairy  Practice. 

KONIG  :   Chemie  der  menschliche  Nahrungs-  und  Genussmittel. 

LEACH  :   Food  Inspection  and  Analysis. 

RICHMOND  :   Dairy  Chemistry. 

ROSENAU  :   Milk  in  its  Relation  to  Public  Health. 

ROTHSCHILD  :   Bibliographia  Lactaria. 

RUSSELL  :   Dairy  Bacteriology. 

SOMMERFELD  :   Haudbuch  der  Milchkunde. 

STOHMAXN  :   Milch-  und  Molkereiproducte. 

SWITHINBANK  and  NEWMAN  :   Bacteriology  of  Milk. 

U.  S.  Dept.  Agriculture,  Farmers'  Bulletins  42  (Facts  about  Milk),  74  (Milk 

as  Food). 

VAN  SLYKE  :    Modern  Methods  of  Testing  Milk  and  Milk  Products. 
WINSLOW  :   Production  arid  Handling  of  Clean  Milk. 
Wisconsin  Agricultural  Experiment  Station,  Bulletins  and  Reports. 

II 

1899.   RICHMOND:   The  Composition  of  Milk  and  Milk  Products.     Analyst, 

24,  197. 

WOODMAN  :   On  the  Determination  of  Added  Water  in  Milk.     J.  Am. 
Chem.  Soc.,  21,  503. 


MILK  367 

1900.  RICHMOND:   The  Composition  of  Milk  and  Milk  Products.     Analyst, 

25,  225. 

WHITAKER  :  The  Milk  Supply  of  Boston  and  Other  New  England 
Cities.  U.  S.  Dept.  Agriculture,  Bureau  of  Animal  Industry, 
Bui.  26. 

1901.  RICHMOND  :   The  Composition  of  Milk.     Analyst,  26,  310. 

1903.  RICHMOND:   The  Composition  of  Milk.     Analyst,  28,  289. 
SHERMAN  :   On  the  Composition  of  Cow's  Milk.     J.  Am.  Chem.  Soc., 

25,  132. 

1904.  LEACH  and  LYTHGOE  :   The  Detection  of  Watered  Milk.     /.  Am. 

Chem.  Soc.,  26,  1195. 

1905.  RICHMOND:   The  Composition  and  Analysis  of  Milk.     Analyst,  30,  325. 

1906.  FREAR  :    American  Milk  and  Milk  Standards.     Proc.  Assn.  State  and 

National  Dairy  and  Food  Departments,  1906,  p.  172. 
LEACH:    Report  on  Dairy  Products  (Refractometer  Test  for  Water- 
ing).    U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  105,  p.  37. 
MULLER  :   Methylene   Blue  as   a  Test  for  the   Freshness  of   Milk. 

Arch.  Hyg.,  56,  108;  Analyst,  31,  299. 
RICHMOND  :   Estimation  of  Fat  in  Homogenized  Milk.     Analyst,  31, 

218,  219-224. 
RICHMOND  and  MILLER  :   Methods  of  Analysis  of  Milk  Used  by  the 

Government  Laboratory.     Analyst,  31,  317. 
ROUSSEAU:    Investigations  on   Sterilization   of   Milk  by  Means  of 

Hydrogen  Peroxide.     Bull.  soc.  pharmacol.,  13,  606  ;  Chem.  Abs.,  1, 

1592. 
SELIGMANN:    (Detection  of  Heated  Milk).     Z.  angew.  Chem.,1906, 

1540. 
SHERMAN  :  Seasonal  Variations  in  the  Composition  of  Cow's  Milk. 

/.  Am.  Chem.  Soc.,  28,  1719. 

1907.  ACKERMANN  :   Refractometric  Detection  of   Added  Water  in  Milk. 

Z.  Nahr.-Genussm.,  13,  186. 
ANDERSON  :   The    Detection  of  Cane   Sugar  in   Milk  and   Cream. 

Analyst,  32,  87. 
BAIER  and  NEUMANN:   Refractometer  Examination   of   Milk.     Z. 

Nahr.-Genussm.,  13,  369. 
DUBOIS:   Analysis  of  Milk  Chocolate.     J.  Am.  Chem.  Soc.,  29,  556. 

1907.  HENKEL  :     Acidity   of   Cows'  Milk.     Milchwirtsch.  ZentrU.,  3,   340  ; 

Chem.  Abs.,  1,  2480. 

HOWARD:   Analysis  of  Ice  Cream.     J.  Am.  Chem.  Soc.,  29,  1622. 
Low :    The  Test  for  Formaldehyde  in  Milk  by  Leach's  Modification 

of  the  Hydrochloric  Acid  and  Ferric  Chloride  Test.     J.  Am. 

Chem.  Soc.,  29,  786. 

1908.  BAIER  and  NEUMANN  :   Detection  of  Calcium  Sucrate  in  Milk  and 

Cream.     Z.  Nahr.-Genussm.,  16,  51. 


368  METHODS   OF   ORGANIC   ANALYSIS 

BURR,  BERBERICH  and  LAUTERWALD  :   Investigations  of  Milk  Serum 

Milchwirtsch.  Zentrbl.,  4,  145,  210,  262;   Chem.  Abs.,  2,  2961. 
FRERICHS  :   Detection  of  Calcium  Sucrate  in  Milk  and  Cream.     Z. 

Nahr.-Genussm.,  16,  682. 
HART  :   Centrifugal  Method  for  Casein  in  Milk.     Wisconsin   Expt. 

Sta.,  Bui.  156  ;  Chem.  Abs.,  2,  675. 
MAI  and  ROTHENFUSSER  :   Detection   of   Added  Water  in  Milk  by 

Means  of  the  Refractometer.     Z.  Nahr.-Genussm.,  16,  7. 

1909.  FENDLER  and  KUHN  :   Determination   of   Dirt  in   Milk.     Z.  Nahr.- 

Genussm.,  17,  513. 

LYTHGOE  and  NURENBERG  :  A  Comparison  of  Methods  for  the  Prep- 
aration of  Milk  Serum.  J.  Ind.  Eng.  Chem.,  1,  38. 

ROBERTSON  :  A  Rapid  Method  of  Determining  the  Percentage  of 
Casein  in  Milk.  /.  Ind.  Eng.  Chem.,  1,  723. 

ROTHENFUSSEH  :  (Nitrate  Test  as  Evidence  of  Added  Water  in 
Milk).  Z.  Nahr.-Genussm.,  18,  353. 

VAN  SLYKE  and  BOSWORTH  :  Volumetric  Method  for  the  Determina- 
tion of  Casein  in  Milk.  J.  Ind.  Eng.  Chem.,  1,  768. 

1910.  AUZINGER  :    (Test  for  Abnormal  Milk).     Milchwirtsch.  Zentrbl.,  5, 

293,  352,  393,  430;  Chem.  Abs.,  4,  619. 
ECKLES  :   Seasonal  Variations  in  Percentages  of  Fat  in  Cows'  Milk. 

Milchwirtsch.  Zentrbl.,  5,  488;   Chem.  Abs.,  4,  620. 
.    HART  :   A  Volumetric  Method  for  the  Estimation  of  Casein  in  Milk. 

J.  Biol.  Chem.,  6, 445. 
LYTHGOE  and  MARSH  :    The  Relation  Between  Fat  and  Calcium  in 

Cream.     J.  Ind.  Eng.  Chem.,  2,  327. 
POETSCHKE  :   The  Determination  of  Sodium  Chloride  in  Milk.     J. 

.Ind.  Eng.  Chem.,  2,  210. 
ROTHENFUSSER  :   Detection  of  Cane  Sugar  and  Calcium  Sucrate  in 

Milk  and  Cream.     Z.  Nahr.-Genussm.,  19,  465. 
TILLMANS:    (Determination  and  Significance  of  Nitrates  in  Milk). 

Z.  Nahr.-Genussm.,  20,  676. 

1911.  BACKE:   Analysis  of  Sweetened  Condensed  Milk.     Analyst,  36,  138. 
BULL:     A   Comparison   Between   the   Refraction   and   the   Specific 

Gravity  of  Milk  Serum  for  the  Detection  of  Added  Water.     J. 
Ind.  Eng.  Chem.,  3,  44. 


CHAPTER   XVIII 
Food  Preservatives1 

FORMALDEHYDE  2 

Detection 

OF  the  many  methods  available  for  the  detection  of  formalde- 
hyde in  milk  and  other  foods  only  three  of  the  best  known  and 
most  delicate  will  be  given  here.  For  other  methods  see  Bui. 
107,  Revised,  Bureau  of  Chemistry,  U.  S.  Department  of  Agri- 
culture. 

Sulphuric  Acid  Test.* — Dilute  2  to  3  cc.  of  milk  with  an 
equal  volume  of  water  in  a  test  tube,  add  carefully,  so  as  not  to 
mix  the  layers,  from  3  to  5  cc.  of  concentrated  commercial  sul- 
phuric acid  or  pure  acid  to  which  a  trace  of  ferric  salt  has  been 
added.  If  formaldehyde  is  present,  a  violet  ring  forms  at  the 
junction  of  the  two  liquids.  The  charring  of  the  milk  by  the 
sulphuric  acid  makes  it  difficult  to  define  the  delicacy  of 
the  test.  One  part  of  formaldehyde  in  100,000  of  milk  can  be 
detected  if  the  milk  is  fresh  and  the  test  is  applied  soon  after 
adding  the  preservative. 

Hydrochloric  Acid  Test.*  —  Mix  10  cc.  of  milk  and  10  cc.  of 
concentrated  hydrochloric  acid  containing  about  2  mg.  of  ferric 
chloride  and  heat  slowly  nearly  to  boiling,  rotating  the  mixture 
occasionally  to  insure  solution  of  the  curd.  In  the  presence  of 

1  Trade  names  and  analyses  of  many  proprietary  preservatives  will  be  found 
in  the  Year-Book  of  the  U.  S.  Department  of  Agriculture  for  1900,  Chapin's 
Theory  and  Practice  of  Infant  Feeding,  and  the  Zeitschrift  fur  die  Untersuchung 
der  Nahrungs-  und  Genussmittel. 

2  See  also  the  methods  for  detection  and  determination  of  f onnaldehyde  given 
in  Chapter  II. 

3  Hehner  :  Analyst,  1896,  21,  95. 

4 Leach:  Ann.  Rpts.  Mass.    State  Board  of   Health,    1897,  558;  1899,  699; 
Food  Inspection  and  Analysis,  p.  140.    See  also  Chapter  II. 
2B  369 


370  METHODS   OF   ORGANIC    ANALYSIS 

formaldehyde  a  violet  color  develops,  otherwise  the  solution 
slowly  turns  brown.  The  test  is  best  performed  in  a  porcelain 
casserole,  and  in  case  of  doubt  the  violet  color  is  made  much  more 
distinct  by  adding  50  to  75  cc.  of  water  after  having  heated  just 
below  boiling  for  about  a  minute.  The  liquid  must  be  observed 
carefully  at  the  moment  of  dilution  as  the  color  brought  out  in 
this  way  fades  very  rapidly.  This  test  is  delicate  to  1  :  250,000, 
but  formaldehyde  added  to  milk  in  such  small  quantities  soon 
disappears.  When  added  to  the  extent  of  1:  50,000  to  1: 100,- 
000  the  presence  of  formaldehyde  in  the  milk  will  be  shown  by 
this  test  for  from  1  to  5  days.1  In  testing  sour  or  stale  milk  the 
brown  color  noted  above  will  often  obscure  the  reaction  given  by 
a  small  amount  of  formaldehyde  until  the  solution  is  diluted  with 
water,  but  at  this  point  the  violet  color  can  be  seen  even  though 
the  milk  may  have  been  much  charred  by  the  acid. 

Grallic  Acid  Test.  —  This  test  has  been  described  in  Chapter 
II.  To  apply  it  to  milk  or  other  liquid  food  acidulate  30  cc.  with 
2  cc.  of  normal  sulphuric  acid  and  distill.  To  the  first  5  cc.  of 
distillate  add  0.2  to  0.3  cc.  of  a  saturated  solution  of  gallic  acid 
in  pure  alcohol,  incline  the  test  tube,  and  pour  in  slowly  3  to  5  cc. 
of  concentrated  sulphuric  acid.  The  presence  of  formaldehyde 
is  shown  by  the  characteristic  blue  ring  described  in  detail  in 
Chapter  II.  In  the  writer's  experience  this  test  is  at  least  twice 
as  delicate  as  either  the  sulphuric  or  the  hydrochloric  acid  test. 
The  latter  would  be  sufficiently  delicate  for  all  practical  pur- 
poses if  milk  samples  could  always  be  tested  while  fresh,  but 
when  small  amounts  of  formaldehyde  have  been  added  one  or  two 
days  previously,  the  gallic  acid  test  may  show  the  preservative 
where  either  of  the  other  tests  would  fail.  In  a  laboratory  ex- 
periment,2 a  sample  of  milk  which  originally  contained  1:  50,000 
formaldehyde  ceased  to  give  any  reaction  by  the  hydrochloric 
acid  and  ferric  chloride  test  after  five  days,  but  the  distillate 
subsequently  obtained  from  30  cc.  of  this  sample  gave  an  un- 
mistakable formaldehyde  reaction  when  tested  with  gallic  acid. 

iEivas:    University    of  Pennsylvania    Medical    Bulletin,    1904,    17,   175. 
Williams  and  Sherman:  J.  Am.  Chem.  Soc.,  1905,  27,  1497. 
2  Williams  and  Sherman:  J.  Am.  Chem.  Soc.t  27,  1499. 


FOOD    PRESERVATIVES  371 

This  gallic  acid  reaction  may  also  be  used  very  satisfactorily 
as  a  means  of  confirming  any  doubtful  results  obtained  by 
either  of  the  preceding  tests,  as  there  is  little  danger  of  inter- 
ference due  to  charring  or  to  the  appearance  of  other  colors. 

Determination 

Very  small  amounts  of  formaldehyde  in  milk  can  be  deter- 
mined by  the  following  method,  which  is  essentially  that  of 
Smith,1  except  that  a  larger  quantity  of  sample  is  used. 

To  300  cc.  of  milk  in  a  round-bottomed  flask  of  about  one 
liter  capacity  add  3  cc.  of  (1  :  3)  sulphuric  acid  and  some  glass 
beads  to  prevent  bumping,  heat  gradually  to  boiling,  preferably 
by  means  of  a  small  rose-top  burner,  and  distill  until  the  dis- 
tillate measures  60  cc.  Transfer  this  to  a  100-cc.  flask,  add  10 
cc.  of  standard  potassium  cyanide  solution,  approximately 
tenth-normal,  and  mix  ;  add  a  mixture  of  15  cc.  of  tenth-nor- 
mal silver  nitrate  and  6  to  8  drops  of  50  per  cent  nitric  acid, 
fill  to  the  mark,  shake,  and  filter  through  dry  paper.  Deter- 
mine the  excess  of  silver  by  the  Volhard  method  and  calculate 
the  results  as  explained  in  Chapter  II.  under  the  description 
of  the  cyanide  method  as  used  for  commercial  solutions  of 
formaldehyde.  The  precautions  and  the  directions  for  stand- 
ardizing there  given  should  also  be  noted. 

By  this  method  from  32  to  39  per  cent  of  the  formaldehyde 
in  the  milk  is  recovered  and  determined  in  the  distillate. 
Assuming  that  the  amount  recovered  represents  35  per  cent,  of 
the  quantity  in  the  milk,  the  latter  can  be  estimated  with  a 
probable  error  of  about  one  tenth.  Using  this  as  a  means  of 
studying  the  disappearance  of  formaldehyde  in  milk,  it  was 
found  in  a  typical  experiment  in  which  the  proportion  added 
was  1:  40,000,  that  nearly  three  fourths  of  the  preservative  had 
disappeared  after  two  days  at  room  temperature.  After  four 
days  no  formaldehyde  was  shown  by  this  method,  but  the  violet 
color  on  dilution  with  water  after  heating  with  hydrochloric 
acid  containing  ferric  chloride  was  unmistakable.  The  latter 
reaction  could  still  be  obtained  after  the  mixture  had  stood  for 
1  J.  Am.  Chem.  Soc.,  1903,  25,  1036. 


372  METHODS   OF   ORGANIC    ANALYSIS 

two  weeks.  When  formaldehyde  is  added  to  milk  in  large 
proportion,  1 : 1000  to  1 : 10,000,  as  in  the  preservation  of 
samples  for  analysis  or  reference,  the  rate  of  disappearance  is 
much  slower. 

HYDROGEN  PEROXIDE 

Detection 

Hydrogen  peroxide  in  uncooked  milk  is  easily  detected  by 
adding,  to  10  to  15  cc.  of  the  milk,  2  to  3  drops  of  a  2  per  cent 
aqueous  solution  of  paraphenylene  diamine  hydrochloride.  In  the 
presence  of  hydrogen  peroxide  a  blue  color  appears  either  imme- 
diately upon  shaking  or  after  a  few  minutes,  depending  upon  the 
amount  present.  The  reaction  depends  upon  the  action  of  an 
oxidizing  enzyme  in  the  milk,  and  the  condition  of  the  milk,  there- 
fore, affects  the  delicacy  of  the  test.  According  to  Arnold  and 
Mentzel 1 1  part  in  40,000  can  be  detected.  Under  ordinary  con- 
ditions the  delicacy  is  probably  somewhat  less  than  this.  In 
comparative  tests  made  immediately  after  adding  the  same 
amounts  of  peroxide  to  sweet  milks  1  to  2  days  old  and  to  very 
sour  curdled  milks  3  to  4  days  old,  the  former  were  found  to 
give  the  reaction  much  more  strongly  than  the  latter.  In 
practice,  however,  the  preservative  would  be  added  while  the 
milk  was  sweet  and  would  probably  disappear  entirely  before 
the  occurrence  of  curdling.  Milk  which  has  been  boiled  can  be 
tested  after  adding  an  equal  volume  of  fresh  milk  known  to  be 
free  from  peroxide. 

Determination 

Chick,2  in  an  investigation  of  the  germicidal  properties  and 
rate  of  disappearance  of  hydrogen  peroxide  in   milk,    used   a 
method  based  upon  the  titration  of  the  iodine  liberated  by  the 
peroxide  on  adding  potassium  iodide  and  sulphuric  acid. 
2  KI  +  H202  +  H2S04  =  K2S04  +  I2  +  H2O. 

Mettler3  has  used  the  method  with  satisfactory  results  in  the 
following  modified  form:  To  40  cc.  of  water,  0.5  gram  of 

1Z.  Nahr.-Genussm.,  1903,  6,  306. 

2  CentralUatt  fur  Bacteriologie  und  Parasitenkunde,  II.  Abth.,  1901,  1,  T05. 

3  Thesis  for  the  degree  of  Bachelor  of  Science,  Columbia  University,  1905. 


FOOD    PRESERVATIVES  373 

potassium  iodide,  and  10  cc.  of  12  per  cent  sulphuric  acid  in  a 
glass-stoppered  flask,  add  10  cc.  of  the  milk,  stopper  tight,  and 
allow  to  stand  in  a  cool,  dark  place  for  two  and  one  half  hours. 
In  order  to  guard  against  any  possible  loss  of  iodine  during  this 
time,  use  a  flask  with  flaring  mouth  as  described  in  connection 
with  the  determination  of  the  iodine  number  (Chapter  VIII), 
and  fill  the  gutter  around  the  stopper  with  a  solution  of  potas- 
sium iodide.  Finally  titrate  the  iodine  which  has  been  set  free 
in  the  milk,  using  a  fiftieth-normal  solution  of  sodium  thiosul- 
phate.  In  this  titration  it  is  not  necessary  to  use  starch  as  in- 
dicator, since  the  disappearance  of  the  yellow  color  produced  by 
the  action  of  the  iodine  upon  the  proteins  affords  a  satisfactory 
end  point.  Test  analyses  gave  results  about  3  per  cent  too  low, 
doubtless  because  of  the  absorption  of  iodine  by  the  milk  fat. 
This  source  of  error  can  be  avoided  by  curdling  the  milk  with 
acid,  filtering,  and  adding  the  iodide  to  a  measured  amount  of 
filtrate;  but  this  is  considered  inadvisable  in  -view  of  the  fact 
that  the  peroxide  may  be  undergoing  decomposition  during  the 
filtration. 

BORIC  ACID  AND  BORATES 

In  routine  milk  analysis 1  the  ash  obtained  in  the  usual  way  is 
treated  with  two  drops  of  dilute  hydrochloric  acid  and  about  a 
cubic  centimeter  of  water.  A  strip  of  turmeric  paper  is  then 
placed  in  the  dish,  allowed  to  soak  for  a  minute,  removed,  and 
allowed  to  dry  in  the  air.  A  deep  red  color  changing  to  green 
or  blue  when  treated  with  dilute  alkali  shows  the  presence  of 
boric  acid.  According  to  Leach  this  reaction  is  delicate  to  1 
part  in  8000.  The  well-known  flame  test  with  methyl  alcohol 
is  less  delicate,  but  can  be  used  in  confirmation. 

The  methods  adopted  by  the  Association  of  Official  Agricul- 
tural Chemists  are  as  follows:2 

Qualitative  Detection 

Render  decidedly  alkaline  with  lime  water  about  25  grams  of  the  sample 
and  evaporate  to  dryness  on  a  water  bath.  Ignite  the  residue  -to  destroy 

1  Leach  :  Food  Inspection  and  Analysis,  2d  ed.,  p.  184. 

2  U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  107,  Revised. 


374  METHODS   OF   ORGANIC    ANALYSIS 

organic  matter.  Digest  with  about  15  cc.  of  water,  add  hydrochloric  acid,  drop 
by  drop,  until  all  is  dissolved,  and  add  1  cc.  in  excess.  Moisten  a  piece  of 
delicate  turmeric  paper  with  the  solution ;  if  borax  or  boric  acid  is  present,  the 
paper  on  drying  will  acquire  a  peculiar  red  color,  which  is  changed  by  ammo- 
nium hydroxide  to  a  dark  blue-green,  but  is  restored  by  acid. 

A  preliminary  test  may  be  made  by  immersing  a  strip  of  turmeric  paper 
in  about  100  cc.  of  liquid  foods,  to  which  about  7  cc.  of  concentrated  hydro- 
chloric acid  has  been  added.  Solid  and  pasty  foods  may  be  heated  with 
enough  water  to  make  them  thoroughly  fluid,  hydrochloric  acid  added  in 
about  the  proportion  of  1  to  13,  and  tested  in  the  same  manner. 

Quantitative  Estimation 

Render  100  grams  of  the  sample  decidedly  alkaline  with  sodium  hydroxide 
and  evaporate  to  dryness  in  a  platinum  dish.  Ignite  the  residue  thoroughly, 
heat  with  about  20  cc.  of  water,  and  add  hydrochloric  acid  drop  by  dropr 
until  all  is  dissolved.  Transfer  to  a  100-cc.  flask,  the  volume  not  being  allowed 
to  exceed  50  to  60  cc.  Add  0.5  gram  of  calcium  chloride  and  a  few  drops  of 
phenolphthalein,  then  a  ten  per  cent  solution  of  caustic  soda  until  a  permanent 
slightly  pink  color  is  produced,  and  finally  add  25  cc.  of  limewater.  Make 
the  volume  up  to  100  cc.  Mix  and  filter  through  a  dry  filter.  To  50  cc.  of 
the  filtrate  add  normal  sulphuric  acid  until  the  pink  color  disappears,  then 
methyl  orange,  and  continue  the  addition  of  the  acid  until  the  yellow  is  just 
changed  to  pink.  Boil  to  expel  carbon  dioxide.  Add  fifth-normal  caustic 
soda  until  the  liquid  assumes  the  yellow  tinge,  excess  of  soda  being  avoided. 
Cool  the  solution,  add  a  little  phenolphthalein  and  an  equal  volume  of  glycerin. 
Titrate  with  standardized  sodium  hydroxide  until  a  permanent  pink  color  is 
produced. 

One  cubic  centimeter  of  fifth-normal  soda  solution  is  equal  to  0.0124  gram 
of  crystallized  boric  acid. 

Low 1  proposes  the  following  modification  of  the  turmeric  test. 
Ten  grams  of  the  sample  (hashed  meat  for  instance)  are  mixed 
with  5  cc.  of  half-normal  solution  of  sodium  carbonate,  dried,  and 
heated  until  volatile  matter  is  completely  driven  off.  The 
charred  mass  is  powdered  and  treated  with  10  cc.  of  water  and 
1  cc.  of  strong  hydrochloric  acid.  After  testing  a  small  portion 
of  the  filtrate  as  usual,  the  remainder  is  placed  in  a  shallow 
dish  with  a  piece  of  turmeric  paper  and  allowed  to  evaporate 
at  40°-50°  in  a  desiccator,  if  necessary  in  a  vacuum,  when  the 
usual  color  should  be  developed. 

1«7.  Am.  Chem.  Soc.t  28,  807. 


FOOD    PRESERVATIVES  375 

This  test  is  said  to  be  much  more  delicate  than  drying  on  a 
steam  bath.  For  full  details  and  discussion  of  delicacy  see  the 
original  paper.  In  this  same  paper,  Low  gives  an  improved 
quantitative  method  for  the  determination  of  boric  acid  in  food, 
and  data  on  the  occurrence  of  boric  acid  in  common  salt  and 
in  laboratory  apparatus  and  reagents. 

FLUORIDES 

If  food  containing  a  small  amount  of  fluoride  is  burned  to 
ash  in  the  usual  way,  the  fluorine  is  likely  to  be  almost  entirely 
lost.  In  the  presence  of  a  considerable  excess  of  alkali  this  loss 
of  fluorine  does  not  occur. 

To  detect  or  determine  fluorides,  add  1  gram  of  sodium  car- 
bonate to  100  cc.  of  milk,1  evaporate,  and  burn  to  ash.  If  only 
qualitative  results  are  required,  examine  for  fluorides  by  the 
well-known  etching  test  on  glass.  For  a  quantitative  deter- 
mination of  the  fluorine,  leach  the  mixture  of  ash  and  sodium 
carbonate  thoroughly  with  hot  water,  nearly  neutralize  with 
sulphuric  acid,  leaving  the  solution  slightly  alkaline,  and  then 
apply  Rose's  method  as  modified  by  Treadwell  and  Koch,  Z. 
anal.  Chem.,  1904,  43,  469. 

The  details  of  the  methods  adopted  by  the  Association  of 
Official  Agricultural  Chemists  are  as  follows  :  2 

Modified  Method  of  Blarez 

Thoroughly  mix  the  sample  and  heat  150  cc.  to  boiling  (in  the  case  of 
solid  foods  the  filtrate  prepared  as  directed  under  salicylic  acid  may  be 
employed).  Add  to  the  boiling  liquor  5  cc.  of  a  10  per  cent  solution  of 
potassium  sulphate  and  10  cc.  of  a  10  per  cent  solution  of  barium  acetate. 
Collect  the  precipitate  in  a  compact  mass  (a  centrifuge  may  be  used  advan- 
tageously) and  wash  upon  a  small  filter.  Transfer  to  a  platinum  crucible 
and  ash. 

Prepare  a  glass  plate  (preferably  of  the  thin  variety  commonly  used  for 
lantern  slide  covers)  as  follows :  First  thoroughly  clean,  polish,  and  coat  on 
one  side  by  carefully  dipping  the  plate  while  hot  in  a  mixture  of  equal  parts 
of  Carnaiiba  wax  and  paraffin.  Near  the  middle  of  the  plate  make  a  distinc- 

1  Or  equivalent  amount  of  other  food. 

2  U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  107,  Revised. 


376  METHODS    OF   ORGANIC    ANALYSIS 

tive  mark  through  the  wax  with  a  sharp  instrument,  such  as  a  pointed  piece 
of  wood  or  ivory,  which  will  remove  the  wax  and  expose  the  glass  without 
scratching  the  latter. 

Add  a  few  drops  of  concentrated  sulphuric  acid  to  the  residue  in  the  cru. 
cible  and  cover  with  the  waxed  plate,  having  the  mark  nearly  over  the  cen- 
ter and  making  sure  that  the  crucible  is  firmly  embedded  in  the  wax.  Place 
in  close  contact  with  the  top  or  unwaxed  surface  of  the  plate  a  cooling  device, 
consisting  of  a  glass  tube  considerably  larger  in  diameter  than  the  crucible, 
the  bottom  of  the  tube  being  covered  tightly  with  a  thin  sheet  of  pure  rub- 
ber. A  constant  stream  of  cold  water  is  passed  through  the  tube.  Heat  the 
crucible  for  an  hour  at  as  high  a  temperature  as  practicable  without  melting 
the  wax  (an  electric  stove  gives  the  most  satisfactory  form  of  heat). 

Remove  the  glass  plate  and  indicate  the  location  of  the  distinguishing 
mark  on  the  unwaxed  surface  of  the  plate  by  means  of  gummed  strips  of 
paper,  then  melt  off  the  wax  by  heat  or  a  jet  of  steam,  and  thoroughly  clean 
the  glass  with  a  soft  cloth.  If  fluorine  be  present,  a  distinct  etching  will  be 
apparent  on  the  glass  where  it  was  exposed. 

Second  Method 

If  it  is  desired,  the  preceding  method  may  be  varied  by  mixing  a  small 
amount  of  precipitated  silica  with  the  precipitated  calcium  fluoride  and 
applying  the  method  given  below  for  the  detection  of  fluosilicates. 

This  method  is  of  value  in  the  presence  of  foods  whose  ash  contains  a 
considerable  amount  of  silica,  which  unites  with  fluorine  and  forms  fluosili- 
cates. The  sulphuric  acid  then  liberates  hydrofluosilicic  acid,  which  would 
escape  detection  by  the  Blarez  modified  method. 

FLUOBORATES  AND  FLUOSILICATES 

(Methods  of  the  Association  of  Official  Agricultural  Chem- 
ists. U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  107,  Revised.) 

Make  about  200  grams  of  the  sample  alkaline  with  lime  water,  evaporate 
to  dryness,  and  incinerate.  Extract  the  crude  ash  first  obtained  with  water, 
to  which  sufficient  acetic  acid  has  been  added  to  decompose  carbonates, 
filter,  burn  the  insoluble  portion,  extract  with  dilute  acetic  acid,  and  again 
filter.  The  insoluble  portion  now  contains  calcium  silicate  and  fluoride, 
while  the  filtrate  will  contain  all  the  boric  acid  present. 

First  Method l 

Incinerate  the  filter  containing  the  insoluble  portion,  mix  with  a  little 
precipitated  silica,  and  place,  with  the  addition  of  1  or  2  cc.  of  concentrated 
sulphuric  acid,  in  a  short  test  tube,  which  is  attached  to  a  small  U-tube  con- 

1  Niviere  and  Hubert,  Moniteur  scientifique,  1895  [4],  9,  324. 


FOOD    PRESERVATIVES  377 

taining  a  few  drops  of  water.  Place  the  test  tube  in  a  beaker  of  water  and 
keep  it  hot  on  the  steam  bath  for  from  30  to  40  minutes.  If  any  fluoride  be 
present,  the  silicon  fluoride  generated  will  be  decomposed  by  the  water  in 
the  U-tube  and  will  form  a  gelatinous  deposit  on  the  walls  of  the  tube. 

Now  test  the  filtrate  as  directed  under  boric  acid.  If  both  hydrofluoric 
and  boric  acids  be  present,  it  is  probable  that  they  are  combined  as  boro- 
fluoride.  If,  however,  silicon  fluoride  is  detected  and  not  boric  acid,  the 
operation  is  repeated  without  the  introduction  of  the  silica,  in  which  case 
the  formation  of  the  silicon  skeleton  is  conclusive  evidence  of  the  presence 
of  fluosilicate. 

Second  Method 

Incinerate  the  filter  containing  the  insoluble  portion  in  a  platinum  cru- 
cible, mix  with  a  little  precipitated  silica,  and  add  1  cc.  of  concentrated  sul- 
phuric acid.  Cover  the  crucible  with  a  watch  glass,  to  the  under  side  of 
which  a  drop  of  water  is  suspended,  and  heat  an  hour  at  the  temperature  of 
70°  to  80°  C.1  The  silicon  fluoride  which  is  formed  is  decomposed  by  the 
water,  leaving  a  gelatinous  deposit  of  silica  and  etching  a  ring  at  the  pe- 
riphery of  the  drop  of  water.  Test  the  filtrate  for  boric  acid  as  described 
above. 

SULPHUROUS  ACID 

The  methods  adopted  by  the  Association  of  Official  Agri- 
cultural Chemists  are  as  follows : 

Qualitative  Detection  2 

To  about  25  grams  of  the  sample  (with  the  addition  of  water,  if  necessary), 
placed  in  a  200-cc.  Erlenmeyer  flask,  add  some  sulphur -free  zinc  and  several 
cubic  centimeters  of  hydrochloric  acid.  In  the  presence  of  sulphites  hydro- 
gen sulphide  will  be  generated  and  may  be  tested  for  with  lead  paper.  Traces 
of  metallic  sulphides  are  occasionally  present  in  vegetables,  and  the  above 
test  will  indicate  sulphites.  Hence  positive  results  obtained  by  this  method 
should  be  verified  by  the  distillation  method. 

It  is  always  advisable  to  make  the  quantitative  determination  of  sulphites, 
owing  to  the  danger  that  the  test  may  be  due  to  traces  of  sulphides.  A  trace 
is  not  to  be  considered  sufficient  indication  of  the  presence  of  sulphur  dioxide 
either  as  a  bleaching  agent  or  as  a  preservative. 

Quantitative  Distillation  Method  8 

Distill  from  20  to  100  grams  of  the  sample  (adding  recently  boiled  water, 
if  necessary)  in  a  current  of  carbon  dioxide,  after  the  addition  of  about  5  cc. 

1  The  watch  glass  may  be  kept  cool  by  means  of  a  piece  of  ice. 

2  U.  S.  Dept.  Agriculture,  Bur.  Chein.,  Bui.  107,  Revised. 

3  Ibid.,  Bui.  137,  p.  116. 


378  METHODS    OF   ORGANIC    ANALYSIS 

of  a  20  per  cent  solution  of  glacial  phosphoric  acid,  until  150  cc.  have  passed 
over.  Collect  the  distillate  in  about  100  cc.  of  nearly  saturated  bromine 
water.  Allow  the  end  of  the  condenser  to  dip  below  the  surface  of  the  liquid 
in  the  receiver.  The  method  and  apparatus  may  be  simplified  without  mate- 
rial loss  in  accuracy  by  omitting  the  current  of  carbon  dioxide,  adding  10  cc. 
of  phosphoric  acid  instead  of  5  cc.,  and  dropping  into  the  distilling  flask  a 
piece  of  sodium  bicarbonate  weighing  not  more  than  a  gram,  immediately 
before  attaching  the  condenser.  The  carbon  dioxide  liberated  is  not  sufficient 
to  expel  the  air  entirely  from  the  apparatus,  but  will  prevent  oxidation  to  a 
large  extent.  When  the  distillation  is  finished,,  boil  off  the  excess  of  bro- 
mine, dilute  the  solution  to  about  250  cc.,  add  5  cc.  of  hydrochloric  acid 
(1  part  of  the  concentrated  acid  to  3  of  water),  heat  to  boiling,  and  precipi- 
tate the  sulphuric  acid  with  a  10  per  cent  solution  of  barium  chloride.  Boil 
for  a  few  minutes  longer,  allow  to  stand  overnight  in  a  warm  place,  filter  on 
a  weighed  Gooch  crucible,  wash  with  hot  water,  ignite  at  a  dull  red  heat, 
and  weigh  as  barium  sulphate. 

Horne1  suggests  that  in  distilling  sulphurous  acid  it  be 
passed  through  cadmium  chloride  solution  to  remove  hydrogen 
sulphide  before  it  reaches  the  bromine  or  iodine  solution. 

For  other  methods  of  detecting  and  determining  sulphurous 
acid  and  sulphites,  as  well  as  for  discussion  and  interpretation, 
see  references  at  the  end  of  this  chapter. 

SALICYLIC  ACID 

The  method  and  precautionary  notes  of  the  Association  of 
Official  Agricultural  Chemists  are  as  follows:2 

A  small  amount  of  salicylic  acid  occurs  naturally  in  many  fruits,  and  not 
more  than  50  grams  should  be  used  for  its  qualitative  detection  in  the  ex- 
amination of  foods.  A  reaction  obtained  with  this  amount  is  due  to  added 
salicylic  acid.  The  method  described  below  is  intended  for  the  quantitative 
determination  of  salicylic  acid.  If  only  a  qualitative  determination  be  de- 
sired, many  of  the  details  may  be  omitted. 

If  the  material  be  a  solid  or  semisolid,  macerate  the  sample  in  a  mortar 
with  water  made  slightly  alkaline,  and  strain  through  a  cotton  bag  or  sepa- 
rate by  means  of  a  centrifuge.  If  preferred,  macerate  from  200  to  300  grams 
with  about  400  cc.  of  water,  and  use  aliquots  of  the  filtrate  for  the  deter- 
mination of  preservatives. 

In  quantitative  work  place  the  macerated  mass  in  a  graduated  flask,  make 
up  to  a  definite  volume  with  water,  and  shake  from  time  to  time  until  solu- 

1  U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  105,  p.  125. 

2  Ibid.,  Bui.  107,  Revised. 


FOOD    PRESERVATIVES  379 

tion  is  complete.  Then  strain  as  directed  above  and  use  an  aliquot  of  the 
nitrate  for  extraction. 

Extract  in  a  separatory  funnel  100  cc.  of  the  sample  or  of  the  aqueous 
solution  prepared  from  the  sample  as  described  above  with  a  sufficient 
amount  of  sulphuric  ether  1  to  prevent  emulsion  after  the  addition  of  2  or 
3  cc.  of  dilute  (1-3)  sulphuric  acid.  Separate  the  clear  aqueous  solution, 
and  if  any  emulsion  is  present,  give  the  separatory  funnel  a  quick,  vigorous 
shake,  and  allow  to  settle  again.  If  the  emulsion  is  not  broken  up  in  this 
way,  it  may  be  accomplished  by  means  of  a  centrifuge,  or  by  adding  10  or 
15  cc.  of  low  boiling  point  gasoline  or  petroleum  ether,  and  shaking  again. 

Separate  the  clear,  aqueous  portion  obtained  from  the  emulsion  and  add 
it  to  the  first  aqueous  portion  separated.  Then  pour  the  ether  into  another 
separatory  funnel,  care  being  taken  that  none  of  the  aqueous  portion  is  left 
with  the  ether.  Return  the  aqueous  portion  to  the  separatory  funnel  and 
again  extract  with  ether,  following  the  same  procedure  as  before.  Repeat 
this  operation  twice  again,  four  separate  extractions  with  ether  being  made 
in  all. 

In  case  of  special  difficulty  in  breaking  up  the  emulsion  in  any  of  the 

1  If  the  nature  of  the  substance  is  such  that  extraction  with  organic  solvents 
is  not  practicable,  as  in  the  case  of  the  presence  of  a  large  amount  of  fat,  the 
salicylic  acid  may  first  be  separated  by  distillation.  In  such  cases  acidify  the 
macerated  material  with  phosphoric  acid  and  transfer  to  a  distilling  flask  with  a 
very  short  neck  and  wide  mouth.  An  Erlenmeyer  flask  with  inside  diameter  of 
mouth  of  1|  inches  is  a  good  shape.  The  tube  connecting  the  flask  with  con- 
denser should  be  very  short,  with  an  inside  diameter  of  not  less  than  f  of  an 
inch. 

Conduct  steam  through  a  small  tube  passing  through  the  stopper  and  dipping 
deeply  into  the  material  in  the  flask.  The  distillation  of  the  salicylic  acid  is 
facilitated  by  submerging  the  distilling  flask  almost  to  the  stopper  in  an  oil 
bath  and  distilling  with  the  temperature  of  the  oil  at  from  120°  to  130°  C. ,  or  by 
adding  about  20  grams  of  sodium  chloride  to  the  contents  of  the  flask  for  each 
100  cc.  of  the  substance,  to  raise  the  boiling  point.  Care  must  be  taken  not  to 
let  the  contents  of  the  flask  get  too  low,  as  the  heat  will  decompose  the  organic 
matter. 

Collect  at  least  600  cc.  of  the  distillate  and  continue  the  distillation  until  the 
last  200  cc.  gives  no  color  on  the  addition  of  a  drop  of  ferric  solution.  The  dis- 
tilling apparatus  should  in  all  cases  be  tested  with  known  amounts  of  salicylic 
acid  in  order  to  determine  the  amount  of  distillate  necessary  to  carry  over  a 
definite  weight  of  salicylic  acid. 

It  is  sometimes  practicable  to  determine  the  salicylic  acid  directly  in  the  dis- 
tillate by  the  colorimetric  method  with  ferric  chloride  given  above.  If  the  min- 
eral acid  used  in  the  distillation  be  carried  over  mechanically,  however,  the 
accuracy  of  the  method  is  greatly  impaired.  Salicylic  acid  may  be  recovered 
from  the  distillate  after  making  alkaline  and  evaporating,  if  desired,  by  extrac- 
tion with  ether  and  estimating  colorimetrically  as  directed  above. 


380  METHODS    OF   ORGANIC    ANALYSIS 

extractions,  a  small  amount  of  ether  may  be  allowed  to  remain  with  the 
aqueous  portion  rather  than  the  reverse,  as  it  is  removed  in  successive  ex- 
tractions. Wash  the  combined  ether  extracts  by  shaking  in  a  separatory 
funnel  with  one  tenth  their  volume  of  water  (using,  however,  not  less  than 
20  cc.  of  water  at  each  washing).  Care  must  be  taken  at  each  washing  to 
separate  the  aqueous  portion  completely  from  the  ether,  but  none  of  the 
ether  should  be  allowed  to  run  into  the  wash  water. 

Distill  slowly  the  greater  part  of  the  ether,  transfer  the  remainder  to  a 
porcelain  dish,  and  allow  to  evaporate  spontaneously.  Thoroughly  dry  in  a 
vacuum  desiccator1  over  sulphuric  acid,  extract  the  dry  residue  with  ten 
portions  of  10  or  15  cc.  each  of  carbon  bisulphide  or  low  boiling  point  petro- 
leum ether,  rubbing  the  contents  of  the  dish  with  a  glass  rod  or  other  suit- 
able instrument  and  transferring  the  successive  portions  of  solvent  to  a 
second  porcelain  dish.  The  extracted  residue  should  finally  be  tested  with 
a  drop  of  ferric-alum  solution,  and  if  any  reaction  for  salicylic  acid  be  given 
it  should  be  taken  up  in  water,  reextracted  with  ether,  and  the  operation 
repeated.  The  gasoline  extract  is  finally  allowed  to  evaporate  spontane- 
ously. 

Dissolve  the  residue  in  a  small  amount  of  hot  water  and  dilute  to  a  defi- 
nite volume.  Dilute  aliquots  of  the  solution  and  match,  in  Nessler  tubes  or 
with  a  colorimeter,  the  color  obtained  by  adding  a  few  drops  of  ferric  chlo- 
ride or  ferric  alum  solution  with  that  of  a  standard  solution  of  salicylic  acid 
containing  about  1  mg.  of  salicylic  acid  in  50  cc.  A  0.5  per  cent  solution  of 
ferric  chloride  should  be  used,  or  a  2  per  cent  solution  of  ferric  alum.2  In 
either  case,  and  especially  with  ferric  chlorid,  an  excess  of  reagent  should 
be  avoided,  although  an  excess  of  0.5  cc.  of  2  per  cent  ferric  alum  solution 
may  be  added  to  50  cc.  of  the  solution  of  salicylic  acid  without  impairing 
the  results. 

Salicylic  acid  may  often  be  separated  from  fat  extracted  with  the  ether 
by  washing  the  ether  solution  with  dilute  ammonium  hydroxide.  Then 
evaporate  the  aqueous  liquid  almost  to  dryness  and  test  with  ferric  solution. 

1  In  examining  a  substance  whose  ether  extract  does  not  give  a  color  or  pre- 
cipitate with  ferric  solution,  the  drying  of  the  residue  and  its  extraction  with 
gasoline  may  be  omitted.     The  residue  may  then  be  transferred  by  means  of 
warm  water  directly  from  the  distilling  flask  to  the  graduated  flask,  in  which  it 
is  made  up  to  a  definite  volume.     Substances  interfering  with  the  ferric  reaction 
may  often  be  removed  by  precipitation  with  ferric  chloride  or  lime. 

2  This  solution  should  be  boiled  until  a  precipitate  appears,  allowed  to  'settle, 
and  filtered.     The  acidity  of  the  solution  is  slightly  increased  in  this  manner, 
but  so  precipitated  it  keeps  clear  for  a  considerable  time,  and  the  turbidity 
caused  by  its  dilution  with  water  is  much  less  and  does  not  appear  for  a  much 
longer  time  than  if  the  unboiled  solution  is  employed.     This  turbidity  is  espe- 
cially objectionable  in  the  quantitative  estimation  of  salicylic  acid,  as  it  interferes 
with  the  exact  matching  of  the  color. 


FOOD    PRESERVATIVES  381 

In  the  case  of  foods  which  yield  to  the  gasoline  solution  of  the  ether  resi- 
due a  color  that  obscures  the  ferric  chloride  reaction  (for  example,  tomatoes), 
the  ether  solution  may  be  evaporated,  the  residue  dried  in  a  desiccator  or  in 
a  current  of  dry  air,  sublimed,  and  collected  on  a  watch  glass  cooled  with 
ice.  Then  dissolve  the  sublimate  in  hot  water  and  test  with  ferric  alum. 

The  same  difficulty  may  often  be  avoided,  and  in  fact  the  extraction  with 
gasoline  of  the  dry  residue  from  the  ether  extraction  may  sometimes  be  ob- 
viated, by  precipitating  before  extraction  with  ferric  chloride  or  calcium 
chloride,  making  alkaline,  and  filtering.  By  this  means  tannin  is  entirely 
separated  from  the  product,  and  other  substances  whose  color  masks  the  sali- 
cylic acid  reaction  are  often  removed. 

Delicacy  of  the  Ferric  Chloride  Test.  —  Using  fresh  1  per 
cent  ferric  chloride  as  reagent  the  test  is  delicate  in  our  hands 
to  a  dilution  of  about  1  :  400,000  when  applied  to  10  c6.  of 
solution,  about  1  :  750,000  to  1  :  1,000,000  if  25  cc.  of  solution 
be  tested.  The  violet  color  obtained  with  such  small  amounts 
of  salicylic  acid  must  be  observed  quickly,  as  it  fades  rapidly, 
passing  through  a  rose-red  color.  A  faint  rose  color  may  also  be 
obtained  on  addition  of  ferric  chloride  to  solutions  containing 
salicylic  acid  in  amounts  too  small  to  show  violet  reaction. 

Interpretation.  —  The  formation  of  a  violet  color  with  ferric 
chloride  is  a  reaction  by  no  means  confined  to  salicylic  acid. 
Mulliken's  tables1  include  many  colorless  compounds  which 
give  more  or  less  distinctly  violet  reactions  with  ferric  chloride, 
and  some  of  these  also  resemble  salicylic  acid  in  solubilities  and 
even  in  volatility.  It  is  not  safe  to  assume  in  testing  foods  that 
a  constituent  volatile  with  steam,  soluble  in  ether,  capable  of 
sublimation  and  crystallization,  and  giving  a  violet  reaction  with 
ferric  chloride,  is  necessarily  salicylic  acid. 

Brand  2  found  that  an  extract  of  caramel  malt  yielded  a  sub- 
stance not  salicylic  acid  which  showed  all  of  these  properties. 
This  substance  he  called  "  maltol." 

The  same  or  similar  interfering  substances  have  been  found  in 
dark  beers  3  and  in  solid  foods  consisting  partly  of  baked  cereal 
products. 4 

1  Identification  of  Pure  Organic  Compounds. 
2Z.  ges.  Brauw.,  15,  303  ;  and  Ber.,  27,  806. 

8  Abraham  :  J.  de  Pharm.  de  Liege,  1898,  5, 173  ;  Z.  Nahr.-Genussm.,  1,  157. 
4  Backer  Ann.  de  falsifications,  Nov.,  1909.     Sherman:  J.  Ind.  Eng.  Chem., 
2,  24.  Backe:  Compt.  rend.,  150,  540  ;  151,  78. 


382  METHODS  OF  ORGANIC  ANALYSIS 

Among  the  tests  for  salicylic  acid,  other  than  the  ferric 
chloride  reaction,  are  the  formation  of  the  methyl  ester  or  the 
nitro-compound,  the  reactions  with  bromine  water  and  with 
Millons's  reagent,  and  the  Jorissen  test. 

The  adoption  by  Mulliken  of  the  methyl-ester  and  nitration 
tests  for  the  identification  of  salicylic  acid  is  sufficient  evidence 
of  their  value  for  cases  in  which  enough  salicylic  acid  is  involved 
to  make  them  available;  but  these  tests  and  also  the  test  with 
bromine  water  seem  not  to  be  sufficiently  delicate  for  the  detec- 
tion of  very  small  amounts. 

The  Millon  and  Jorissen  tests,  however,  are  very  delicate, 
and  should  be  commonly  used  to  confirm  the  findings  of  the 
official  ferric  chloride  test. 

Test  with  Millori s  Reagent l 

To  10  to  20  cc.  of  the  final  aqueous  solution  to  be  tested,  add 
2  drops  of  Millon's  reagent  (prepared  as  described  in  Chapter 
XV),  mix  by  shaking,  and  immerse  in  boiling  water  for  45 
minutes  unless  a  sufficient  color  develops  in  a  shorter  time. 
In  the  presence  of  salicylic  acid  a  red  or  pink  color  is  obtained. 

By  heating,  if  necessary,  for  as  long  as  45  minutes,  this  test  is 
made  so  delicate  that  with  practice  and  with  blank  tests  for 
comparison  no  difficulty  was  found  in  detecting  the  presence 
of  1  part  salicylic  acid  in  2,000,000  of  water  when  20  cc.  were 
tested;  when  only  10  cc.  were  tested,  the  pinkish  tint  was 
barely  perceptible  at  this  dilution.  Longer  heating  and  varia- 
tions in  the  amount  of  reagent  added  were  tried  without  appre- 
ciably altering  the  result.  The  limit  of  delicacy  of  the  test 
with  Millon's  reagent  as  here  used  seems,  therefore,  to  be 
reached  by  heating  in  boiling  water  for  45  minutes  and  to  lie 
at  a  dilution  of  about  1:2,000,000. 

The  Millon  reaction  also  has  the  advantage  over  the  ferric 
chloride  test  that  the  color  produced  even  with  very  small 
amounts  of  salicylic  acid  shows  no  evidence  of  fading  on  stand- 
ing overnight;  but  on  account  of  the  large  number  of  sub- 

1  Sherman  and  Gross  :  J.  Ind.  Eng.  Chem.,  3,  492. 


FOOD    PRESERVATIVES  383 

stances  which  respond  to  the  Millon  reagent 1  it  seems  unlikely 
that  this  reaction  will  prove  as  useful  as  that  of  Jorissen. 

Jorissen  Test2 

This  test  in  its  original  form  is  as  follows : 

To  the  solution  to  be  tested  add  4  or  5  drops  of  a  10  per  cent 
solution  of  potassium  (or  sodium)  nitrite,  4  or  5  drops  of  acetic 
acid,  1  drop  of  a  10  per  cent  solution  of  copper  sulphate,  and 
heat  to  boiling.  In  the  presence  of  salicylic  acid  the  solution 
turns  reddish  and  with  more  than  a  very  minute  amount  be- 
comes blood-red.  Jorissen  found  that  phenol  behaved  in  the 
same  way,  but  benzoic  acid  did  not.  Abraham  found  that 
maltol  does  not  give  this  reaction  and  recommended  it  as  the 
most  reliable  test  for  salicylic  acid. 

By  diminishing  the  amount  of  copper  used  in  the  above 
directions  and  prolonging  the  heating,  this  test  can  be  made 
much  more  delicate  than  at  first  reported  and  considerably 
more  delicate  than  the  ferric  chloride '  reaction.  The  longer 
heating  is  necessary  to  fully  develop  the  characteristic  color,  at 
least  when  only  very  small  amounts  of  salicylic  acid  are  present, 
and  the  reduction  in  the  amount  of  copper  diminishes  the  slight 
green  color  due  to  the  reagent  which  otherwise  may  interfere 
with  the  more  delicate  tests. 

The  modified  Jorissen  test  as  now  used  for  very  small 
amounts  of  salicylic  acid  is  as  follows : 3  Bring  the  solution  to 
be  tested  into  a  test  tube,  add  4-5  drops  of  10  per  cent  sodium 
or  potassium  nitrite,  4-5  drops  of  50  per  cent  acetic  acid,  and 
1  drop  of  one  per  cent  copper  sulphate.  Shake  after  addition 
of  each  reagent  and  finally  place  in  a  boiling  water  bath  in 
such  a  position  that  the  test  liquid  is  completely  immersed  in 
the  boiling  water  and  allow  to  stand  for  45  minutes,  then 

1  Vaubel:  Z.  angew.   Chem.,  1900,  1125.     Nasse:  Pfluger's.  Archiv  f.  d.  ges. 
Physiol.,  83,  361    (1901).    Mann:  Physiological  Histology,   pp.    321-323,    and 
Chemistry  of  the  Proteids,  p.  7. 

2  Jorissen  :  Bulletins  de  I"1  Academic  Royal  des  Sciences,  etc.,  Belgique,  3d 
series,  3,  259.     Sherman:  J.  Ind.  Eng.  Chem.,  2,  24.     Sherman  and  Gross: 
Ibid.,  3,  492. 

3  Sherman  and  Gross  :  J.  Ind.  Eng.  Chem.,  3,  492. 


384          METHODS  OF  ORGANIC  ANALYSIS 

remove,  allow  to  cool,  and  examine  against  a  white  background, 
viewing  the  tube  both  vertically  and  horizontally  and  compar- 
ing with  a  blank  test  in  which  the  same  amounts  of  reagents 
have  been  added  to  pure  water. 

In  this  way,  the  presence  of  as  little  as  0.005  to  0.01  milli- 
gram of  salicylic  acid  in  pure  water  solution  can  be  detected. 
Faint  but  perceptible  reactions  were  obtained  with  5  to  8  cc.  of 
a  solution  of  1  :  1,000,000  and  with  18  to  25  cc.  of  solutions  of 
1  :  3,000,000  to  1  :  3,500,000. 

No  advantage  has  been  found  in  a  brine  bath  over  a  water 
bath,  in  longer  heating  than  45  minutes,  nor  in  varying  the 
amounts  of  nitrite  and  acetic  acid  used.  When  larger  amounts 
of  salicylic  acid  are  present,  a  drop  of  stronger  copper  sulphate 
solution  may  be  used,  up  to  a  10  per  cent  solution  as  originally 
recommended.  Except  with  very  small  amounts  of  salicylic 
acid  the  red  color  of  the  Jorissen  reaction  develops  quickly  on 
heating,  and  the  long  immersion  in  the  water  bath  then  becomes 
unnecessary  if  only  qualitative  results  are  required. 

A  feature  which  will  be  of  great  importance  in  colorimetric 
estimations  of  small  amounts  of  salicylic  acid  is  that  while  the 
violet  color  of  the  ferric  chloride  test  fades  rapidly,  the  red 
color  of  the  Jorissen  test  is  quite  stable.  Even  the  faint  colors 
obtained  by  long  heating,  where  only  very  minute  amounts  of 
salicylic  acid  are  involved,  have  shown  no  deterioration  when, 
allowed  to  stand  overnight. 

It  may  also  be  noted  that  the  ferric  chloride  and  Jorissen. 
tests  may  be  applied  to  the  same  portion  of  solution.  After 
making  the  ferric  chloride  test  the  solution  is  cautiously  diluted 
with  water  until  the  violet  color  just  disappears  and  then  very 
carefully  submitted  to  the  modified  Jorissen  test,  when  if  salicylic 
acid  is  present  a  pink  color  will  appear. 

Maltol,  isomaltol,  orcin,  arbutin,  resorcin,  phlprizin,  and 
methyl-ethyl-aceto-acetate  (all  of  which  are  among  the  sub- 
stances giving  blue,  violet,  or  violet-red  colors  with  ferric 
chloride)  do  not  respond  to  the  Jorissen  reaction. 

Phenol  gives  about  the  same  color  as  salicylic  acid  in  both 
the  Millon  and  the  Jorissen  tests,  but  the  limits  of  delicacy  are 


FOOD    PRESERVATIVES  385 

quite  different.  Phenol  can  be  detected  by  the  Millon  reaction 
to  about  1  :  2,000,000.  In  the  Jorissen  test,  phenol  1  :  100,000 
gives  practically  the  same  color  as  salicylic  acid  1  : 1,000,000. 

Saligenin  gives,  in  the  Jorissen  reaction,  a  red  color  at 
1:10,000;  a  yellowish  tint  at  1:100,000;  no .  reaction  at 
1 : 1,000,000.  The  limit  of  delicacy  for  the  ferric  chloride  re- 
action with  saligenin  lies  between  1  :  10,000  and  1  :  20,000. 

2-oxy-isophthalic  acid  gives  the  Jorissen  reaction  up  to  a 
dilution  of  1  :  100,000  but  is  easily  distinguished  from  salicylic 
acid  in  the  color  which  it  gives  with  ferric  chloride. 

BENZOIC  ACID  AND  BENZOATES 

The  methods  of  the  Association  of  Official  Agricultural 
Chemists l  are  as  follows  : 

QUALITATIVE  DETECTION 

Separate  benzole  acid  as  directed  for  salicylic  acid.  If  benzoic  acid  be 
present  in  considerable  quantity,  it  will  crystallize  from  the  evaporated  ether 
in  shining  leaflets  with  characteristic  odor  on  heating.  Dissolve  the  residue 
in  hot  water,  divide  into  two  portions  (a)  and  (6),  and  test  by  the  following 
methods : 

(1)    First  Method 

Make  portion  (a)  alkaline  with  ammonium  hydroxide,  expel  the  excess  of 
ammonia  by  evaporation,  take  up  the  residue  with  water,  and  add  a  few 
drops  of  a  neutral  0.5  per  cent  solution  of  ferric  chloride.  The  presence  of 
benzoic  acid  will  be  indicated  by  the  formation  of  a  brownish  colored  pre- 
cipitate of  ferric  benzoate. 

(2)    Second  Method  (Mahler's  Method  Modified) 

Add  to  the  water  solution  (portion  5),  prepared  as  described  above,  from 
1  to  3  cc.  of  third-normal  sodium  hydroxide  and  evaporate  to  dryness.  To  the 
residue,  add  5  to  10  drops  of  concentrated  sulphuric  acid  and  a  small  crystal 
of  potassium  nitrate.  Heat  for  10  minutes  in  glycerol  bath  at  120°  to  130°  C., 
or  for  20  minutes  in  a  boiling  water  bath.  This  causes  the  formation  of 
meta-di-nitro-benzoic  acid.  In  no  case  must  the  temperature  exceed  130°  C. 
After  cooling,  add  1  cc.  of  water,  and  make  decidedly  ammoniacal ;  boil  the 
solution,  to  break  up  any  ammonium  nitrite  which  may  have  been  formed. 
Cool  and  add  a  drop  of  fresh  colorless  ammonium  sulphide,  without  allowing 
the  layers  to  mix.  A  red-brown  ring  indicates  benzoic  acid.  This  is  due  to 

1  U.  S.  Dept.  Agriculture,  Bur.  Chem.,Bul.  107,  Revised,  p.  181;  and  Bui.  137, 
pp.  110-112,  113,  117-118. 

2c 


386  METHODS   OF   ORGANIC    ANALYSIS 

the  formation  of  ammonium  rneta-di-amido-benzoic  acid.  On  mixing,  the 
color  diffuses  through  the  whole  liquid;  on  heating  it  finally  changes  to 
greenish  yellow,  owing  to  the  decomposition  of  the  amido  acid.  This  fur- 
nishes a  means  of  distinguishing  benzoic  acid  from  salicylic  or  cinnamic 
acids.  Both  the  latter  form  amido  compounds,  which  are  not  destroyed  by 
heating.  The  presence  of  phenolphthalein  interferes  with  this  test. 

QUANTITATIVE  ESTIMATION 

General  Method  of  Preparation 

Grind  in  a  sausage  machine,  if  solid  or  semisolid,  thoroughly  mix  the  sample, 
and  transfer  a  convenient  quantity  (about  150  grams)  to  a  500-cc.  graduated 
flask.  Add  enough  pulverized  sodium  chloride  to  saturate  the  water  in  the 
sample,  render  alkaline  with  sodium  hydroxide  or  milk  of  lime,  and  dilute 
to  the  mark  with  a  saturated  salt  solution.  Allow  to  stand  for  at  least  two 
hours  with  frequent  shaking,  and  filter.  If  the  sample  contains  large  amounts 
of  matter  precipitable  by  salt  solution,  it  is  advisable  to  follow  a  method 
similar  to  that  given  under  "  Salt  or  dried  fish."  When  alcohol  is  present, 
follow  the  method  given  under  "  Cider  and  similar  products  containing  al- 
cohol." Where  large  amounts  of  fats  are  present,  it  is  well  to  make  an  alka- 
line extraction  of  the  filtrate  before  proceeding  as  directed  under  Extraction 
and  Titration.  The  following  will  illustrate  the  manner  of  applying  the 
method  to  various  classes  of  food  products : 

Special  Methods  of  Preparation 

Ketchup.  —  To  150  grams  of  the  sample  add  15  grams  of  pulverized  sodium 
chloride  and  transfer  the  mixture  to  a  500-cc.  graduated  flask,  using  about 
150  cc.  of  a  saturated  solution  of  sodium  chloride  for  rinsing.  Make  slightly 
alkaline  to  litmus  paper  with  strong  sodium  hydroxide  and  complete  the 
dilution  to  500  cc.  with  saturated  salt  solution.  Allow  to  stand  at  least 
two  hours  with  frequent  shaking  and  then  filter  through  a  large  folded 
filter.  If  any  difficulty  is  experienced,  the  mixture  may  be  centrif  uged  or 
squeezed  through  a  muslin  bag  before  filtering. 

Jellies,  Jams,  Preserves  and  Marmalades.  —  Dissolve  150  grams  of  the  sample 
in  about  150  cc.  of  saturated  salt  solution  and  add  15  grams  of  pulverized 
sodium  chloride.  Render  alkaline  to  litmus  paper  with  milk  of  lime. 
Transfer  to  a  500-cc.  graduated  flask  and  dilute  to  the  mark  with  saturated 
salt  solution.  Allow  to  stand  at  least  two  hours  with  frequent  shaking, 
centrifuge  if  necessary,  and  filter  through  a  large  folded  filter. 

Cider  and  Similar  Products  containing  Alcohol.  —  Render  250  cc.  of  the  sample 
alkaline  to  litmus  paper  with  sodium  hydroxide  and  evaporate  on  the  steam 
bath  to  about  100  cc.  Transfer  the  sample  to  a  250-cc.  flask,  add  30  grams 
of  pulverized  sodium  chloride,  and  shake  until  dissolved.  Dilute  to  the 


FOOD    PRESERVATIVES  387 

original  volume,  250  cc.,  with  saturated  salt  solution,  allow  to  stand  at  least 
two  hours  with  frequent  shaking,  and  filter  through  a  folded  filter. 

Salt  or  Dried  Fish.  —  Transfer  50  grams  of  the  ground  sample  to  a  500-cc. 
flask  with  water.  Make  slightly  alkaline  to  litmus  paper  with  strong  sodium 
hydroxide  and  dilute  to  the  mark  with  water.  Allow  to  stand  at  least  two 
hours  with  frequent  shaking  and  then  filter  through  a  folded  filter.  Pipette 
accurately  as  large  a  portion  of  the  filtrate  as  possible  (at  least  300  cc.)  into 
a  second  500-cc.  flask.  Add  30  grams  of  pulverized  sodium  chloride  for  each 
100  cc.  of  solution.  Shake  until  the  salt  has  dissolved  and  dilute  to  the  mark 
with  saturated  salt  solution.  Mix  thoroughly  and  filter  off  the  precipitated 
protein  matter  on  a  folded  filter. 

Extraction  and  Titration 

Pipette  a  convenient  portion  of  the  filtrate  (100  to  200  cc.),  obtained  as 
above,  into  a  separatory  funnel.  Neutralize  the  solution  to  litmus  paper 
with  hydrochloric  acid  (1 :  3)  and  add  an  excess  of  5  cc.  of  the  same  acid. 
In  the  case  of  salt  fish  a  precipitation  of  protein  matter  usually  occurs  on 
acidifying,  but  the  precipitate  does  not  interfere  with  the  extraction.  Ex- 
tract carefully  with  chloroform,  using  successive  portions  of  70,  50,  40,  and 
30  cc.  To  avoid  emulsion  shake  each  time  cautiously  (vigorous  shaking  is 
not  necessary).  The  chloroform  layer  usually  separates  readily  at  the  bottom 
of  the  funnel  after  standing  a  few  minutes.  If  any  emulsion  forms,  it  can 
be  broken  up  by  stirring  the  chloroform  layer  with  a  glass  rod.  If  this  is 
unsuccessful,  the  emulsified  portion  may  be  drawn  off  into  a  second  funnel 
and  given  one  or  two  sharp  shakes  from  one  end  of  the  funnel  to  the  other. 
If  this  also  fails,  the  emulsion  should  be  centrif uged  for  a  few  moments.  As 
this  is  a  progressive  extraction  great  care  must  be  taken  to  draw  off  as  much 
of  the  clear  chloroform  solution  as  possible  after  each  extraction,  but  under 
no  circumstances  must  any  of  the  emulsion  be  drawn  off  with  the  chloroform 
layer.  If  care  is  taken  not  to  draw  off  any  of  the  emulsion,  it  is  unnecessary 
to  wash  the  chloroform  extract. 

Transfer  the  combined  chloroform  extract  to  a  porcelain  dish,  rinsing  the 
container  several  times  with  a  few  cubic  centimeters  of  chloroform,  and 
evaporate  to  dryness  at  room  temperature  in  a  current  of  dry  air.  (See  note.) 
Dry  the  residue  overnight  (or  until  no  odor  of  acetic  acid  can  be  detected  in 
case  the  product  is  a  ketchup)  in  a  sulphuric  acid  desiccator.  Dissolve  the 
residue  of  benzoic  acid  in  neutral  alcohol  (30  to  50  cc.),  add  about  one  fourth 
this  volume  of  water,  a  drop  or  two  of  phenolphthalein  solution,  and  titrate 
with  twentieth-normal  sodium  hydroxide ;  1  cc.  of  twentieth-normal  sodium 
hydroxide  =  0.0072  gram  anhydrous  sodium  benzoate. 

Note.  —  If  a  blast  is  convenient,  it  is  preferable  to  evaporate  the  whole  ex- 
tract at  room  temperature.  For  this  purpose  the  following  simple  apparatus 
may  be  used  :  A  wide-mouth  salt  bottle  is  fitted  with  a  cork ;  a  glass  tube 
extends  through  the  center  of  the  cork  to  the  bottom  of  the  bottle,  and  its 


388  METHODS   OF   ORGANIC    ANALYSIS 

upper  end  is  attached  to  the  blast  by  a  rubber  tube.  As  many  other  glass 
tubes  as  convenient  are  passed  through  the  cork  around  the  central  tube. 
These  terminate  just  inside  the  cork,  and  outside  the  cork  are  bent  outward 
and  downward  at  an  angle  of  about  45°  C.  The  bottle  is  filled  with  calcium 
chlorid  and  by  this  means  a  current  of  dry  air  can  be  delivered  to  the  dish 
containing  the  extract.  In  the  absence  of  a  blast  an  electric  fan  may  be  used 
for  evaporating  the  extract. 

If  it  is  impracticable  to  evaporate  the  chloroform  spontaneously  or  by 
means  of  a  blast  it  maybe  transferred  from  the  separatory  funnel  to  a300-cc. 
Erlenmeyer  flask,  rinsing  the  separatory  funnel  three  times  with  5  or  10  cc. 
of  chloroform.  Distill  very  carefully  to  about  one-fifth  the  original  volume, 
keeping  the  temperature  down  so  that  the  chloroform  comes  over  in  drops, 
not  in  a  steady  stream.  Then  transfer  the  extract  to  a  porcelain  evaporating 
dish,  rinsing  the  flask  three  times  with  5  or  10  cc.  portions  of  chloroform  and 
evaporate  to  dryness  spontaneously. 

SACCHAKIN 

The  usual  method  of  testing  for  saccharin  is  to' extract  it  by 
means  of  ether,  then  convert  it  into  sodium  salicylate  by  heat- 
ing with  sodium  hydroxide,  and  finally  apply  the  test  for 
salicylate.  The  sweet  taste  of  the  saccharin  serves  as  a  pre- 
liminary test  for  its  presence  in  the  ether  extract. 

The  recent  ruling  under  the  Federal  Food  and  Drugs  Act 
against  the  presence  of  saccharin  in  foods  adds  greatly  to  the 
significance  of  its  presence  or  absence.  The  analyst  therefore 
should  not  only  follow  the  details  of  the  official  method  with 
care  in  order  to  avoid  confusing  saccharin  with  "  false  saccharin" 
or  salicylic  acid,  but  should  also  consult  the  original  papers  on 
both  saccharin  and  salicylic  acid  which  are  given  in  the  refer- 
ences at  the  end  of  this  chapter. ' 

The  qualitative  method  and  precautionary  notes  of  the  Asso- 
ciation of  Official  Agricultural  Chemists  are  as  follows : 

Extract  with  ether  (after  maceration  and  exhaustion  with  water,  if  nec- 
essary), as  described  under  salicylic  acid.  Allow  the  ether  extract  to  evap- 
orate spontaneously  and  note  the  taste  of  the  residue.  The  presence  of 
saccharin  to  the  amount  of  20  mg.  per  liter  is  indicated  by  a  sweet  taste. 
This  may  be  confirmed  by  heating  with  sodium  hydroxide,  as  described 
below,  and  detecting  the  salicylic  acid  formed  thereby.  Results  by  this 
method  indicating  the  presence  of  a  faint  trace  of  saccharin  in  wines  which 
did  not  contain  it  have  been  frequently  obtained,  owing  to  the  presence  in 
wine  of  so-called  "  false  saccharin." 


FOOD    PRESERVATIVES  389 

Acidify  50  cc.  of  a  liquid  food  (or  the  aqueous  extract  of  50  grams  of  a 
solid  or  semisolid,  prepared  as  directed  in  the  official  method  for  salicylic 
acid  as  given  above)  and  extract  with  ether.  Test  the  extracted  matter  in 
the  usual  way  for  salicylic  acid,  ^return  the  gasoline  extract  to  the  dish  con- 
taining the  residue,  dilute  the  whole  to  about  10  cc.  volume,  and  add  2  cc. 
of  sulphuric  acid  (1 : 3.)  Bring  the  solution  to  the  boiling  point  and  add  a 
5  per  cent  solution  of  potassium  permanganate,  drop  by  drop,  to  slight 
excess;  partly  cool  the  solution,  dissolve  in  it  a  piece  of  sodium  hydroxide, 
and  filter  the  mixture  into  a  silver  dish  (silver  crucible  lids  are  well 
adapted  to  the  purpose)  ;  evaporate  to  dryness  and  heat  for  20  minutes  at 
210°  to  215°  C.  Dissolve  the  residue  in  water,  acidify  and  extract  with 
ether,  evaporate  the  ether,  and  test  the  residue  with  two  drops  of  a  2  per 
cent  solution  of  ferric  alum.  By  this  method  all  the  so-called  false  saccharin 
and  the  salicylic  acid  naturally  present  (also  added  salicylic  acid  when  not 
present  in  too  large  amount)  are  destroyed,  while  5  mg.  of  saccharin  per 
liter  is  detected  with  certainty. 

BETA-NAPHTHOL  * 

Extract  200  cc.  of  the  sample  (or  of  its  aqueous  extract  prepared  as  on 
page  378)  with  10  cc.  of  chloroform  in  a  separatory  funnel,  add  a  few  drops 
of  alcoholic  potash  to  the  chloroform  extract  in  a  test  tube,  and  place  in  a 
boiling  water  bath  for  two  minutes.  The  presence  of  beta-naphthol  is 
indicated  by  the  formation  of  a  deep  blue  color,  which  changes  through 
green  to  yellow. 

ABRASTOL1 
(Calcium  o-mono-sulphonate  of  ^3-naphthol) 

(a)   Sinibaldi's  Method'2 

Make  50  cc.  of  the  sample  alkaline  with  a  few  drops  of  ammonium  hy- 
droxide and  extract  with  10  cc.  of  amyl  alcohol  (ethyl  alcohol  is  added  if  an 
emulsion  is  formed).  Decant  the  amyl  alcohol,  filter  if  turbid,  and  evap- 
orate to  dryness.  Add  to  the  residue  2  cc.  of  a  mixture  of  equal  parts  of 
strong  nitric  acid  and  water,  heat  on  the  water  bath  until  half  of  the  water 
is  evaporated,  and  transfer  to  a  test  tube  with  the  addition  of  1  cc.  of  water. 
Add  about  0.2  cc.  of  ferrous  sulphate  and  an  excess  of  ammonium  hydroxide, 
drop  by  drop,  with  constant  shaking.  If  the  resultant  precipitate  is  of  a 
reddish  color,  dissolve  it  in  a  few  drops  of  sulphuric  acid,  and  add  ferrous 
sulphate  and  ammonium  hydroxide  as  before.  As  soon  as  a  dark-colored 
or  greenish  precipitate  has  been  obtained,  introduce  5  cc.  of  alcohol,  dissolve 
the  precipitate  in  sulphuric  acid,  and  shake  the  fluid  well  and  filter.  In 

1  The  methods  given  are  those  adopted  by  the  Association  of  Official  Agri- 
cultural Chemists.     U.  S.  Dept.  Agriculture,  Bur.  Chein.,  Bui.  107,  Revised. 

2  Moniteur  scientifique,  1893  (4),  7,  842. 


390  METHODS    OF   ORGANIC   ANALYSIS 

the  absence  of  abrastol  this  method  gives  a  colorless  or  light  yellow  liquid, 
while  a  red  color  is  produced  in  the  presence  of  0.01  gram  of  abrastol. 

(&)   Sangle'-Ferriere's  Method1 

Boil  200  cc.  of  the  sample  with  8  cc.  of  concentrated  hydrochloric  acid 
for  one  hour  in  a  flask  with  a  reflux  condenser  attached.  Abrastol  is  thus 
converted  into  beta-naphthol  and  is  detected  as  directed  above. 

SUCROL   OR   DULCIN2 

(para-phenetol  carbamid) 
(a)  Morpurgo's  Method  3 

Evaporate  about  100  cc.  of  the  sample  (or  of  the  aqueous  extract  pre- 
pared as  directed  on  page  378)  to  a  sirupy  consistency  after  the  addition  of 
about  5  grams  of  lead  carbonate,  and  extract  the  residue  several  times  with 
alcohol  of  about  90  per  cent;  evaporate  the  alcohol  extract  to  dryness; 
extract  the  residue  with  ether,  and  allow  the  ether  to  evaporate  sponta- 
neously in  a  porcelain  dish.  Add  2  or  3  drops  each  of  phenol  and  concen- 
trated sulphuric  acid  and  heat  for  about  5  minutes  on  the  water  bath ;  cool ; 
transfer  to  a  test  tube  and  pour  ammonium  hydroxide  or  sodium  hydroxide 
over  the  surface  with  the  least  possible  mixing.  The  presence  of  dulcin  is 
indicated  by  formation  of  a  blue  zone  at  the  plane  of  contact. 

(&)  Jorissen's  Method4 

Suspend  the  residue  from  the  ether  extract  obtained  as  directed  above  in 
about  5  cc.  of  water ;  add  from  2  to  4  cc.  of  an  approximately  10  per  cent 
solution  of  mercuric  nitrate,  and  heat  from  5  to  10  minutes  on  the  water 
bath.  In  the  presence  of  sucrol  a  violet-blue  color  is  formed,  which  is 
changed  to  a  deep  violet  by  the  addition  of  lead  peroxide. 

In  the  foregoing  selection  of  methods  for  the  detection  of 
food  preservatives,  preference  has  been  given  to  those  which 
have  been  adopted  by  the  Association  of  Official  Agricultural 
Chemists,  whose  methods  are  usually  accepted  as  standard  in 
questions  relating  to  adulteration  of  food.  Other  methods, 
however,  should  not  be  neglected.  It  is  believed  that  the  fol- 
lowing references  will  put  the  reader  in  touch  with  the  most 
important  literature. 

1  Comp.  rend.,  1893,  117,  796. 

2  Method  of  the  Association  of  Official  Agricultural  Chemists  (Joe.  eft.). 

3  Z.  anal.  Chem.,  1896,  35,  104. 

4  Ibid.,  p.  628. 


FOOD    PRESERVATIVES  391 

REFERENCES1 


ALLEN  :    Commercial  Organic  Analysis. 
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II 

1899.   BEYTHIEN  and  HEMPEL  :    (Determination  of  Boric  Acid  and  Borax 
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1901.  PELLET  :   Nature  of  the  Substance  giving  the  Ferric  Chloride  Re- 

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1  These  references  are  to  literature  on  the  occurrence,  detection,  and  deter- 
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uses  and  effects. 


392          METHODS  OF  ORGANIC  ANALYSIS 

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FOOD    PRESERVATIVES  393 

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WOODMAN  and  BURWELL  :  Detection  of  Formic  Acid  in  Food. 
Tech.  Quart.,  21,  1. 


394  METHODS   OF   ORGANIC    ANALYSIS 

ZERBAN  and  NAQUIN:  Determination  of   Sulphurous  Acid   in  Mo- 
lasses.    U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  116. 

1909.  BACKE  :  (Source  of  Error  in  Salicylic  Acid  Test).     Ann.  falsifications, 

Nov.,  1909. 
CARLINFANTI  and  TUFFI  :  Use  and  Detection  of  Fluorides  in  Tomato 

Conserves.     Arch.  farm,   sper.,   8,    377;  Chem.  Zentr.,   1909,  II, 

1765. 
FISCHER  and  GRUENERT:  Detection  of  Benzoic  Acid  in  Meat  and 

Fats.     Z.  Nahr.-Genussm.,  17,  721. 
French   Official    Methods.       Verh.   kais.    Gesundheitsamt.,   32,   1292; 

Chem.  Abs.,  3,  554. 
GENTH  :  Test  for  Saccharin  in  Foods  and  Beverages.     Am.  J.  Pharm., 

81,  536  ;  Chem.  Abs.,  4,  352. 
HILLYER  :    Method  for  determining    Sodium  Benzoate  in  Ketchups 

or  Other  Food  Materials.     J.  Ind.  Eng.  Chem.,  1,  538. 
JORGENSON:  Detection  of  Saccharin  in  Beer.     Ann.  falsifications,  2, 

58. 
LANGE  :   Sulphurous  Acid  in  Gelatine.    Arb.  kais.  Gesundheitsamte,  32, 

144;  Chem.  Abs.,  3,  2989. 
ROBIN:   Test  for  Benzoic   Acid   in   Fatty  Substances.     Ann.  chim. 

anal.,  13,  431 ;   Chem.  Abs.,  3,  1938. 
SCHWARZ  and  WEBER  :   Quantitative  Determination  of  Formic  Acid 

in  Fruit  Sirups.     Z.  Nahr.-Genussm.,  17,  194. 
TESTONI  :   Estimation  of  Saccharin  in  Foods.      Z.  Nahr.-Genussm., 

18,  577. 

U.  S.  Dept.  Agriculture,  Bur.  Chem.,  Bui.  116,  12. 
WAUTERS  :    Detection  of  Saccharin.     /.  Soc.  Chem.  Ind.,  28,  733. 

1910.  BACKE  :    (Substances  mistaken  for  Salicylic  Acid  in  Ferric  Chloride 

Test).     Campt.  rend.,  150,  540;  151,  78. 
BERTAINCHAND  and  GAUVRY  :   Presence  of  Boron  in  Tunisian  Wines. 

Ann.  chim.  anal. ,15,  179;   Chem.  Abs.,  4,  2179. 
BERTRAND  and  AGULHON  :   Determination  of  Boric  Acid.     Bui.  soc. 

chim.,  7,  90,  125;  Chem.  Abs.,  4,  1439. 
CASSEL  :   Estimation  of  Salicylic  Acid  by.the  Distillation  of  its  Dilute 

Aqueous  Solutions.     Chem.  News,  101,  289  ;  Chem.  Abs.,  4,  2426. 
COLLINS:    The  Transfer  of  Boric  Acid  from  Cattle  Food  to  Cows' 

Milk.      Durham  County  Council  Bui,  3,  21 ;    Chem.  Abs.,  4,  1324. 
COMANDUCCI  :   Detection   of   Saccharin.     Boll.  chim.  farm.,  49,  791 ; 

Chem.  Zentrbl.,  1910,  II,  1951. 
DOWZARD  :   Detection  and  Determination  of    Sulphurous   Acid   in 

Lime  Juice.     Am.  J.  Pharm.,  81,  561 ;  Chem.  Abs.,  4,  1348. 
DUGAST  :   Presence  of  Boron  in  the  Wines  of  Algeria.     Compt.  rend., 

150,  839;   Chem.  Abs.,  4,  2974. 
FISCHER  and  GREUNERT  :   Detection  of  Benzoic  Acid  in  Meat  and 

Fats.     Z.  Nahr.-Genussm.,  20,  580. 


FOOD    PRESERVATIVES  395 

GREIBEL  :   Benzole  Acid  in  Cranberries.    Z.  Nahr.-Genussm.,  19,  241. 
HUBERT  :    Disappearance  of  Sulphurous  Acid  (when  added  to  wine). 

Ann.  chim.  anal,  14,  453;   Chem.  Abs.,  4,  1080. 
KICKTON  and  BEHNCKE  :    Occurrence    of    Fluorine    in    Wines.      Z. 

Nahr.-Genussm.,  20,  193. 
KUHN  and  RUHLE  :   Determination   of   Sulphurous  Acid   in  Meats. 

Z.  Nahr.-Genussm.,  20,  10;   Chem.  Abs.,  4,  2851. 
PAWLOWSKI  :   Detection  of  Saccharin  in  Beer.     Z.  ges.  Brauw.,  32, 

281;  Chem.  Abs.,  4,  948. 
PELLET  :   Normal  Presence  of  Salicylic  Acid  in  Wines.     Rev.  soc.  hyg. 

alim.,  5,  806 ;  Chem.  Abs.,  4, 1647. 
PERKIER  :   Presence  of  Formaldehyde  in  Certain  Foods.     Rev.  soc. 

liyg.  alim.,  5,  804;   Chem.  Abs.,  4,  1635. 
BOSSET  :   Detection  of  Fluoride  in  Foods.     Ann.  chim.  anal.,  14,  365  ; 

Chem.  Abs.,  4,  623. 
SHERMAN  :   A  Source   of   Error   in  the   Examination  of   Foods  for 

Salicylic  Acid.     J.  Ind.  Eng.  Chem.,  2,  24. 
VON  DER  HEIDE  and  JAKOB  :    The  Detection  of  Benzoic,  Cinnamic, 

and  Salicylic  Acids  in  Wine.     Z.  Nahr.-Genussm.,  19,  137. 
VON  FELLENBERG  :    Determination  of  Salicylic  Acid  in  Preserves. 

Z.  Nahr.-Genussm.,  20,  63. 
YODER  and  TAGGART  :    Occurrence  of  Formaldehyde  in  Sugar  Cane 

Juice  and  Sugar-House  Products.     J.  Ind.  Eng.  Chem.,  2,  260. 
1911.   FINCKE:   Determination    of    Formic    Acid    in    Foods.      Z.   Nahr.- 
Genussm.,  21,  1. 
FOLIN  and  FLANDERS  :   Determination   of    Benzoic   Acid.      J.   Am. 

Chem.  Soc.,  33,  1622. 
FRANZEN  and  EGGER  :    Quantitative  Determination  of  Formic  Acid. 

J.  prakt.  Chem.,  83,  323. 
LYTHGOE  and  MARSH  :     The  Detection  of   Benzoic  Acid  in  Coffee 

Extract.     /.  Ind.  Eng.  Chem.,  3,  842. 
LOOCK  :   Preservatives  in  Fruit  Juices  with  Special  Reference  to  the 

Detection  of  Formic  Acid.      Z.  offentl.  Chem.,  16,  350;   Chem. 

Abs.,  5.  537. 
POLENSKE  :   Detection  of  Benzoic  Acid  in  Food.     Arb.  kais.  Gesundh., 

38,  149;  Chem.  Abs.,  5,  3860. 
POLENSKE  and  KOPKE  :    Determination  of  Saltpeter  in  Meat.     Arb. 

kais.  Gesundh.,  36,  291 ;   Chem.  Abs.,  5,  1802. 
PRICE  and  INGERSOLL  :     Effects   of    Nitrates   and    Nitrites   on    the 

Turmeric  Test  for  Boric  Acid.     U.  S.  Dept.  Agr.,  Bur.  Chem., 

Bui.  137,  115. 
SHERMAN  and  GROSS  :   The  Detection  of  Salicylic  Acid.    /.  Ind.  Eng. 

Chem.,  3,  492. 
VIERHOUT  :   Quantitive  Estimation  of  Salicylic  Acid  in  Fruit  Juices. 

Z.  Nahr.-Genussm.,  21,  664. 


SUBJECT  INDEX 


Abbe  refractometer,  165. 

Abderhalden  and  Koelker's  optical 
method  for  proteolytic  enzymes, 
(ref.)  332. 

Abraham's  viscosity  method,  183. 

Abrastol,  389,  390,  (ref.)  392-393. 

Absolute  alcohol,  2,  32,  33. 

Acetanilid,  determination  in  vanilla 
extract,  (ref.)  49. 

Acetate,  129,  (ref.)  131-132. 

Acetic  acid,  129,  (ref.)  131-132;  de- 
termination in  calcium  acetate,  129- 
131 ;  in  vinegar,  128. 

Acetin  method  for  glycerol,  279. 

Acetone  determination,  30. 

Acetylizable  impurities  in  glycerin,  283. 

Acetyl  number,  144,  160. 

Acid,  arachidic,  134,  179,  180;  behenic, 
134;  benzoic,  385,  386;  boric,  373, 
374;  butyric,  133,  195;  capric,  133, 
195 ;  caproic,  133,  195 ;  caprylic, 
133 ;  195 ;  carnaubic,  134,  139 ; 
cerotic,  135;  clupanodonic,  137; 
dihydroxystearic,  137,  195;  erucic 
135 ;  hypogseic,  135 ;  isolinolenic, 
137  ;  lanoceric,  138 ;  lauric,  134, 
195;  lignoceric,  134;  linoleic,  136; 
linolenic,  137,  139;  linusic,  137; 
melissic,  135;  myristic,  134,  195; 
oleic,  135,  139,  195,  197;  palmitic, 
134,  195;  phycetoleic,  135;  ricino- 
leic,  138;  sativic,  137;  stearic,  134, 
195;  sulphurous,  377.  (See  also 
Acids.) 

Acid-albumin,  311. 

Acidity,  degrees  of,  147. 

Acidity  of  butter  fat,  194 ;  of  glycerin, 
278;  of  lubricating  oil,  231;  of 
milk,  (ref.)  367. 

Acid  method  for  phosphorus,  304. 

Acid  number,  147. 

Acids,  fatty,  133,  138,  139,  167,  196; 
fatty  and  resin,  271 ;  in  butter  fat, 
139,  195.  (See  also  Acid.) 

Acree's  formaldehyde  color  reaction 
for  proteins,  (ref.)  331.  . 

Added  water,  detection  of,  in  milk, 
363,  364,  (ref.)  366-368. 

Addition  reactions  of  aldehydes,  35;  of 
formaldehyde,  44. 


Adiabatic  calorimeter,   (ref.)  264. 

Adulterants  in  oils,  174-185,  199-202, 
206-210,  213-217. 

Albuminates,  311. 

Albuminoids,  308,  311. 

Albumins,  308,  310. 

Alcohol,  2,  31-33;  absolute,  2,  32,  33; 
aldehyde-free,  172;  cetyl,  169; 
denatured,  4,  32,  33;  density  of 
solutions,  13,  15,  16 ;  detection  of, 
5,  23,  32,  33 ;  determination  of,  6-24, 
31-33;  fusel  oil  in,  28,  32,  33; 
methyl  alcohol  in,  24,  32,  33 ;  myri- 
cyl,  169;  octodecyl,  169;  refer- 
ences, 31-33 ;  standards  of  purity, 
29 ;  TL  S.  P.,  2,  29. 

Alcohol-ether  mixtures,  analysis  of, 
(ref.)  32. 

Alcohols,  1,  31-33,  169.  (See  also 
Alcohol.) 

Alcohol-soluble  proteins,   308,   310. 

Alcoholysis  of  fatty  substances,  (ref.) 
172. 

Aldehydes,  34,  47,  48,  49 ;  detection  of, 
34;  determination  of  in  alcoholic 
liquors,  (ref.)  32.  (See  also  Ben- 
zaldehyde,  Formaldehyde,  Vanillin.) 

Alkali-albumin,  311. 

Alkali  method  for  phosphorus,  303  ; 
for  sulphur,  298. 

Alkalinity  of  vinegar  ash,  123,  125,  126, 
128. 

Allein  and  Gaud  reagent,  78. 

Allen  method  for  peptic  activity,  327; 
for  protein  nitrogen,  (ref.)  330. 

Allihn  method  for  dextrose,  107. 

Allihn's  table  for  dextrose,  108. 

Allspice,  starch  content  of,  112. 

Almond  extract,  determination  of  ben- 
zaldehyde  in,  46,  49. 

Almond  oil,  171. 

Almond  shells,,  starch  content  of,  112. 

Alumina  cream,  89. 

Aluminate  in  soap,  273. 

Ammoniacal  silver  nitrate  test  for 
aldehydes,  34. 

Amyl  alcohol,  28,  32,  33. 

Amylases,  113-120,  (ref.)  121-122. 

Angular  rotation,  83. 

Apple  vinegar,  123. 


397 


398 


SUBJECT   INDEX 


Arabinosazone,  62,  64. 

Arabinose,  50,  54,  63,  85.  (See  also 
Pentosans,  Pentoses.) 

Arachidic  acid,  134,  179,  180,  (ref.) 
199-201. 

Arachis  oil,  171,  179,  184,  (ref.)  199-201. 

Arnold-Wedemeyer  method  for  nitro- 
gen, 289. 

Ash :  in  butter,  186 ;  in  coal,  256,  264  ; 
in  grain  products,  337 ;  in  milk, 
356,  357  ;  in  sugar,  97  ;  in  vinegar,  125. 

Asphaltic  matter  in  oils,  222,  235,  238. 

Atwater  calorimeter,  239. 

Azo-compounds,  determination  of  nitro- 
gen in,  (ref.)  306. 

Balance,  Westphal,  162. 

Balling  hydrometer  ("spindle"),  100. 

Bang  method  for  reducing  sugars, 
(ref.)  86. 

Baobab  oils,  (ref.)  202. 

Bardach's  protein  reaction,  (ref.)  331. 

Barfoed's  method  for  reducing  sugars, 
76. 

Barlow's  method  for  sulphur,  296. 

Bates  polariscope,  (ref.)  86. 

Baudouin  test,  181 ;  influence  of  ran- 
cidity on,  (ref.)  202. 

Baume  hydrometer,  219. 

Baume  scale,  219. 

Beef  tallow,  170. 

Beeswax,  170,  (ref.)  173. 

Beets,  determination  of  sugar  in,  98. 

Behenic  acid,  134. 

Bellier  method  for  arachidic  acid,   181. 

Benedict  method  for  reducing  sugars, 
(ref.)  86. 

Benedict  reagent,  78,  (ref.)  86. 

Benzaldehyde,  46,  47,  49. 

Benzene,  water  in,  (ref.)  238. 

Benzoate,  detection  and  determination, 
385,  386,  (ref.)  393-395. 

Benzoic  acid,  detection  and  determina- 
tion of,  385,  386,  (ref.)  391-395; 
occurrence  of,  in  cranberries,  (ref.) 
392,  395 ;  standardization  of  calo- 
rimeter by  means  of,  242;  titration 
of,  387 ;  vs.  cinnamic  acid  in  food 
analysis,  (ref.)  393. 

Berthelot  bomb  calorimeter,  239. 

Berthelot  method  for  sulphur,  301. 

Beta-naphthol,  389. 

Bicarbonate,  detection  of,  in  milk,  365. 

Bisulphite  reaction  of  aldehydes,  35. 

Biuret  reaction,  313. 

Blarez  method  for  fluorides,  375. 

Blasdale's  viscosity  test,  183. 

Boiling  point,  determination  of,  24. 


Boiling  point  method  for  alcohol,  22. 

Bomb  calorimeter,  239. 

Borates,  in  food,  373,  (ref.)  392-395; 
in  soap,  273. 

Boric  acid,  detection  and  determination, 
373-375,  (ref.)  391-395;  natural  oc- 
currence in  foods,  375,  (ref.)  391- 
395. 

Boron  in  wines,  (ref.)  394. 

Bran,  starch  content  of,  112. 

British  thermal  unit,  241. 

Brix   hydrometers   ("spindles"),    100. 

Bromine   substitution   number,    207. 

Bromine  water  test  for  salicylic  acid, 
382. 

Brugelmann  method  for  sulphur,  296. 

Buckwheat  hulls,  starch  content  of,  112. 

Burning  oils,  (ref.)  236-237.  (See  also 
Illuminating  oils.) 

Burning  point,  233. 

Butter,  185  ;  acid  content  of,  (ref.)  200 ; 
analysis  of,  186 ;  detection  of  coco- 
nut fat,  198,  (ref.)  202 ;  Dutch,  (ref.) 
200,  202  ;  iodine  number,  (ref.)  200 ; 
keeping  qualities  of,  (ref.)  200 ; 
renovated,  (ref.)  200;  standard, 
185;  substitutes,  197-202 ;  synthetic 
color  in,  (ref.)  202 ;  volatile  acid 
content,  195-197,  (ref.)  200;  water 
content,  186-187,  (ref.)  201-202. 

Butter  fat,  analysis  of,  1-88-194;  com- 
position of,  195-197;  references, 
199-202;  relation  of  physical  and 
chemical  characters,  196  ;  separation 
of  acids  in,  (ref.)  139 ;  variations  in 
properties,  195. 

Butyric  acid,  133. 

Butyrin,  146. 

Butyro-refractometer,  165. 

Cacao  butter,  170. 

Calcium  acetate,  12*9. 

Calorific  power,  245 ;  determination  of, 
239 ;  estimated  and  determined  in 
wood,  etc.,  253;  of  coal,  257-265; 
of  fatty  oils,  171 ;  of  fuel  oils  and 
gasoline,  247  ;  of  organic  compounds, 
245;  of  petroleum  oils,  248;  rela- 
tion to  chemical  composition,  257, 
260. 

Calorimeter,  adiabatic,  (ref.)  264;  At- 
water, 239;  Berthelot,  239;  Emer- 
son, 239;  Junker,  263;  Mahler, 
239;  radiation  correction,  244;  ref- 
erences, 261-265. 

Calorimetry,  239-265. 

Cane,  determination  of  sucrose  in,  98. 

Cane  sugar,  see  Sucrose. 


SUBJECT   INDEX 


399 


Cane  sugar  factories,  chemical  control 
of,  (ref.)  104. 

Capric  acid,  133,  195. 

Caproic  acid,  133,  195. 

Caprylic  acid,  133,  195. 

Carbohydrates  (general) ,  50-57 ;  be- 
havior on  oxidation,  58 ;  detection 
of,  by  Molisch  reaction,  57 ;  hy- 
drolysis of,  60,  94,  106,  109,  110; 
reactions  with  acids,  56 ;  reactions 
with  hydrazines,  61 ;  rotation  of 
polarized  light,  78-85 ;  separation 
by  pure  yeasts,  (ref.)  104  ;  separation 
in  grain  products,  340 ;  specific  ro- 
tatory powers,  82-85. 

Carbonate,  in  glycerin,  278 ;  in  milk, 
365  ;  in  soap,  273. 

Carius  method,  (ref.)  307. 

Carnaliba  wax,  170. 

Carnaiibic  acid,  134,  (ref.)  139. 

Casein  determination,  (ref.)  368. 

Casein  test  for  formaldehyde,  39. 

Castor  oil,  141,  142,  171. 

Cellulose,  53,  55,  61. 

Cereals,  determination  of  starch  in, 
106,  110,  112,  (ref.)  121 ;  loss  of 
phosphorus  in  ashing,  (ref.)  307. 

Cereals,  see  Grain  products. 

Cerotic  acid,  135. 

Cetyl  alcohol,  169. 

Chilling  point,  232. 

Chinese  wood  oil,  204,  (ref.)  217. 

Cholesterol,  169,  (ref.)  200;  separa- 
tion from  phytosterol,  (ref.)  201. 

Cider,  composition  of,  (ref.)  132 ;  de- 
termination of  benzoic  acid  in,  386. 

Cider  vinegar,   123-126,   (ref.)    132. 

Cinnamic  acid,  detection  of,  .(ref.) 
393,  395. 

Citral,  determination  of,  (ref.)  49. 

Clarification  in  sugar  analysis,  79,  89, 
90,  92,  (ref.)  105. 

Classon's  method  for  sulphur,  296. 

Clerget  method  for  sucrose,  94. 

Cleveland  cup  tester,  233. 

Cloud  test,  232,  (ref.)  236. 

Cloves,  starch  content  of,  112. 

Clupanodonic  acid,  137. 

Coagulated  proteins,  312. 

Coal,  254-265 ;  accuracy  in  sampling, 
(ref.)  263;  American,  (ref.)  262; 
calorific  power,  254-265 ;  classifica- 
tion, (ref.)  261,  262;  composition  in 
relation  to  calorific  power,  254-260, 
(ref.)  262-265  ;  deterioration  of  sam- 
ples, (ref.)  263;  gases  occluded  in, 
(ref.)  264  ;  influence  of  oxygen  in,  254, 
(ref.)  264 ;  losses  in  storage,  (ref.) 


264 ;  production  of,  in  1910,  (ref.)  265  ; 
proximate  analysis,  256 ;  purchase  on 
specifications,  (ref.)  263-265 ;  sam- 
pling, (ref.)  263,  265;  standard,  for 
gasmaking,  (ref.)  264 ;  sulphur  deter- 
mination, 257 ;  volatile  matter,  256, 
(ref.)  263-265 ;  weathering  of,  (ref.) 
263. 

Coals  of  Illinois,  (ref.)  262. 

Coals  of  the  United  States,  (ref.)  263. 

Coal  tar  oils,  determination  in  other 
oils,  (ref.)  236. 

Cocoa  products,  determination  of 
starch  in,  (ref.)  121. 

Cocoa  shells,  starch  content  of,  112. 

Coconut  fat,  detection  in  butter,  198 ; 
ethyl  ester  number,  (ref.)  202. 

Coconut  oil,  141,  170;  detection  of, 
198,  (ref.)  201-202;  of  high  iodine 
value,  (ref.)  202.  (See  also  Coco- 
nut fat.) 

Codliveroil,  171,  (ref.)  200-201.. 

Coefficient  of  purity,  see  Quotient  of 
purity. 

Cold  test,  232,  (ref.)  236. 

Collagens,  311. 

Colophony,  170,  (ref.)  215: 

Color  reactions  of  proteins,  313-316. 

Colza  oil,  171. 

Combustion,  heat  of,  168,  239,  245,  (ref.) 
261-265. 

Compensator,  81. 

Compressed  oxygen  method  for  sulphur, 
301. 

Condensation  reactions  of  aldehydes, 
42,  46,  48. 

Condensed  milk,  analysis  of  sweetened, 
(ref.)  368. 

"Constants,"  analytical,  of  oils,  fats, 
and  waxes,  143,  170,  171,  176, 
196. 

Copak  oil,  178. 

Copper  as  protein  precipitant,  317. 

Corn  stover,  starch  content  of,  112. 

Cottonseed  meal,  influence  of  feeding, 
on  fat,  (ref.)  200. 

Cottonseed  oil,  171,  177,  178,  184,  208, 
(ref.)  199-202. 

Cottonseed  stearin,  170. 

Coumarin,  determination  in  vanilla  ex- 
tract, (ref.)  49. 

Cream,  267,  268. 

Creosote,  characteristics  of,  (ref.)  217. 

Crismer's  test,  194. 

Crude  fiber,  337. 

Crude  petroleum,  218,  (ref.)  237.  (See 
also  Petroleum.) 

Cyanide  method  for  formaldehyde,  44. 


400 


SUBJECT   INDEX 


Cylinder  oils,  (ref .)  236. 
Cylinder  stock,  221. 

Defren's  method  for  reducing  sugars,  74. 
Degrees  of  acidity  (fats),  147. 
Denatured  alcohol,  4. 
Density :    of   alcohol   solutions,    13,    15, 

16 ;     of    sugar    solutions,     100 ;     of 

water,  17. 

Detergents,  see  Soap. 
Dextrin,  53,  61,  85. 
Dextrose,    51 ;      detection,    62-68,    77 ; 

determination,  70,  74,  107 ;   reducing 

power,  69,  73,   75 ;    rotating  power, 

84,  85,  (ref.)  105. 
Diabetic  foods,  (ref.)  348. 
Diabetic    sugar,    51.     (See    also    Dex- 
trose.) 

Diastase,  see  Amylases. 
Diastase   method   for   determination   of 

starch,  110. 
Diastatic    power,    113-120,    (ref.)    121- 

122. 
Dichromate    method    for    alcohol,    23 ; 

for  glycerol,  284. 

Dichromate  solution  as  light  filter,  93. 
Dinitrobenzoate  test  for  alcohol,  5. 
Dioxystearic  acid,  137,  195. 
Diphenyl     hydrazine     as     reagent     for 

sugars,  63. 

Dirt  in  milk,  (ref.)  368. 
Disaccharides,  50,  51. 
Distillation  method  for  alcohol,  8 ;    for 

gasoline,  249 ;    for  petroleum,  220. 
Distilled  vinegar,  124. 
Drying  oils,  203. 
Dry  lead  clarification  of  sugar  solutions, 

90,  (ref.)  104. 
Dulcin,  390. 

Dulong's  formula,  252,  254. 
Dyer  method  for  nitrogen,  291. 

Ebullioscope,  22. 

Edestin,  339. 

Edible  oils  and  fats,  174. 

Elaidin  test,  136. 

Elastins,  311. 

Emerson  calorimeter,  239,   (ref.)  263. 

Engler  distillation  test,  220. 

Engler  flask,  220.     . 

Engler  viscosimeter,  226,  (ref.)  237,  238. 

Enzymes,     amylolytic,     113-120,     (ref.) 

121-122  ;   proteolytic,  323-328,  (ref.) 

329,  331-333. 
Erucic  acid,  135. 

Eschka  method  for  sulphur,  257. 
Essential  oils,  aldehydes  and  ketones  in, 

(ref.)  48,  49. 


Ester  number,  147. 

Esters,  glyceryl,  143 ;  in  alcoholic 
liquors,  (ref.)  32 ;  in  wood  alcohol, 
31 ;  saponification  data  of,  146. 

Ether-alcohol  mixture,  analysis  of,  (ref.) 
32. 

Ether  extract,  see  Fat. 

Ethyl  ester  number,  (ref.)  202. 

Evaporation  test  for  oils,  235. 

Factories,  cane  sugar,  chemical  control, 
(ref.)  104. 

Fat,  analysis,  143-217 ;  determination, 
268,  335,  354,  358.  (See  also  Butter, 
Olive  Oil,  etc.) 

Fats,  140-217;  acetyl  number,  160; 
alcohols  of,  169  ;  analytical  methods, 
143  ;  analytical  properties,  170,  171 ; 
characteristics  of  animal,  169,  (ref.) 
172,  173;  classification,  141;  "con- 
stants" of,  143,  170-171;  edible, 
174 ;  heat  of  combustion,  168,  171  ; 
index  of  refraction,  164,  170,  171, 
176,  196,  211;  iodine  number, 
148-157,  170,  171,  176,  197,  211; 
Maumene  number,  157,  171,  176, 
211;  melting  point,  144,  167,  170; 
solubilities,  142 ;  specific  gravity, 
162,  170,  171. 

Fatty  acids,  133-139,  167.  (See  also 
Butter,  Fats,  etc.) 

Fatty  and  resin  acids  in  soap,  269. 

Fatty  oils  in  lubricants,  225. 

Fehling's  method  for  reducing  sugars,  70. 

Fehling's  solution,  69,  70. 

Ferric  acetate  as  protein  precipitant,  317. 

Ferric  chloride  test  for  salicylic  acid,  381. 

Fish  liver  oil,  (ref.)  201. 

Fish  oil,  137,  139,  205,  208,  217. 

Fixed  carbon  in  coal,  257. 

Flashing  point,  233,  (ref.)  237. 

Flour  ;  analysis  of,  334-340  ;  composition 
of,  344-346;  starch  content  of,  112. 

Fluoborates,  376. 

Fluorides,  375,  (ref.)  392-395. 

Fluorine,  see  Fluorides. 

Fluosilicates,  376. 

Folin's  method  for  sulphur,  (ref.)  307. 

Food  preservatives,  369-395. 

Foods,  see  under  names  of  individual 
articles  of  food. 

Formaldehyde,  36 ;  detection  of,  38-40, 
369-371,  (ref.)  391-395;  deter- 
mination, 40-45,  48,  49,  371,  (ref.) 
392,  393;  influence  of,  on  detection 
of  hydrogen  peroxide  in  milk,  (ref.) 
393 ;  occurrence  in  foods,  (ref.) 
392-395. 


SUBJECT   INDEX 


401 


Formic  acid,  detection  and  determina- 
tion in  foods,  (ref.)  394-395. 

Friction  tests  on  lubricants,  234,  (ref.) 
237. 

Fructose,  see  Levulose. 

Fruit  juices,  (ref.)  395. 

Fruit  sugar,  see  Levulose. 

Fuchsin  test  for  aldehydes,  35. 

Fuels,  239-265.     (See  also  Coal,  etc.) 

Furfural,  32,  57. 

Fusel  oil,  28,  32,  33. 

Galactans,  53,  59,  61. 

Galactosazone,  62,  64. 

Galactose,  51,  59,  62,  64,  84,  85. 

Gallic  acid  test  for  formaldehyde,  39. 

Gas-making,  valuation  of  oils  for, 
(ref.)  236. 

Gasoline,  237,  247,  250. 

Gelatin,  311. 

Gerard  reagent,  78. 

Gliadin,  339,  (ref.)  346-348. 

Globulins,  308,  310. 

Glucosazone,  62,  64-69. 

Glucose,  see  Dextrose. 

Glucose  vinegar,  124. 

Glutelins,  308. 

Glutenin,  339. 

Glycerides,  142-143.     (See  also  Fats.) 

Glycerin,  276-287.     (See  also  Glycerol.) 

Glycerol,  1,  140,  275,  283;  determina- 
tion by  acetin  method,  279 ;  by 
dichromate  method,  284 ;  in  soap, 
270,  287 ;  significance  in  vinegar, 
(ref.)  132. 

Glyceryl  esters,  143. 

Glycogen,  53,  54,  61,  85. 

Glycoproteins,  309,  312. 

Grain  products,  334-348 ;  analysis  of, 
334-342  ;  composition,  344-346  ;  defi- 
nitions and  standards,  343 ;  diges- 
tibility and  nutritive  value,  346; 
references,  346-348. 

Grain  vinegar,  124. 

Grape  sugar,  see  Dextrose. 

Grape  vinegar,  123. 

Graphite,  deflocculated,  (ref.)  236. 

Gray's  method  for  crude  petroleum,  221. 

Greases,  lubricating,  235. 

Gunning  method  for  nitrogen,  289,  291. 

Half-shade  polariscope,  80,  81. 

Halogens,  as  protein  precipitants,  316, 
319 ;  determination  in  organic  sub- 
stances, (ref.)  306. 

Halphen's  reaction,  177,  (ref.)  199-201. 

Hanus  method  for  iodine  number,  153, 
156. 

2D 


Heat  capacity  of  calorimeter,  240. 

Heat  coagulation  of  proteins,  316. 

Heat  of  combustion,  239-245  ;  adiabatic 
method,  (ref.)  263;  of  fats  and 
fatty  oils,  168,  171;  of  fuels,  245- 
265 ;  of  other  organic  substances, 
(ref.)  261. 

Heavy  distillate,  221. 

Heavy  metals  as  protein  precipitants, 
316. 

Hehner  number,  144,  148,  191. 

Hemicellulose,  61. 

Hemoglobins,  309. 

Hemp  oil,  171. 

Herzfeld's  method  for  reducing  sugars, 
96. 

Hexabromide  test,  209,  214. 

Histones,  308. 

Homogenized  milk,  (ref.)  367. 

Honey,  (ref.)  105. 

Hopkins  distilling  head,  292. 

Hubl  number,  148,  156,  170,  171. 

Hydrazine  reactions  of  sugars,  61. 

Hydrazones,  determination  of  nitro- 
gen in,  (ref.)  306. 

Hydrochloric-acid-casein  test  for  for- 
maldehyde, 39. 

Hydrogen  peroxide,  detection  and  de- 
termination, 372,  (ref.)  391-393; 
method  for  formaldehyde,  41. 

Hydrolysis   of    carbohydrates,    60. 

Hydrometer,  Baume,  219. 

Hydrothermal  equivalent  of  calorimeter, 
241. 

Hydroxy  acids,  137,  138,  161. 

Hypogseic  acid,  135. 

Ice  cream,  analysis  of,  (ref.)  367. 
Illuminating  oils,  218,   221,    (ref.)   236- 

238. 
Immersion     refractometer,     17-21,     32, 

33. 

Immiscible  solvents,  224. 
Imported   sugars  and   molasses,   testing 

of,  (ref.)  104. 
Index    of    refraction,     17-22,     164-166. 

(See  also  under  Fats.) 
International    methods,    for   oil   testing, 

(ref.)  237;   for  sugar  analysis,  91. 
Invertase    method    for     sucrose,     (ref.) 

105. 

Invert  sugar,  51,  85. 
lodimetric    method    for     formaldehyde, 

40. 
Iodine     number,     148-157.     (See     also 

Fats.) 

lodoform  test  for  alcohol,  5. 
Isolinolenic  acid,  137. 


402 


SUBJECT   INDEX 


Japan  wax,  170. 

Jodlbauer  method  for  nitrogen,  290. 

Jorissen    method    for    sucrol    or    dulcin, 

390. 

Jorissen  reaction  for  salicylic  acid,  382. 
Junker  gas  calorimeter,  (ref.)  263. 

Kapok  oil,  178,  202. 

Kendall    method   for    reducing    sugars, 

(ref.)  86. 
Keratins,  311. 
Kjeldahl     method     for    nitrogen,    288- 

295,  306-307. 

Kjeldahl-Wilfarth  method,  289. 
Koettstorfer  number,  144. 

Lactobiose,  see  Lactose. 

Lactometer,  353. 

Lactosazone,  62,  64. 

Lactose,  51,  59,  61,  64;  determination, 
59,  70,  74,  361;  hydrolysis  of,  52, 
104  ;  rotating  power,  84,  85  ;  separa- 
tion from  maltose,  (ref.)  103 ;  solu- 
bility, 54,  55. 

Lanoceric  acid,  138. 

Lard,  170,  (ref.)  199-202;  effect  of 
feed  upon  properties,  (ref.)  202. 

Lard  oil,  141,  171,  182,  184. 

Laurent  polariscope,  80. 

Laurie  acid,  134,  195. 

"Lead  number"  of  maple  products, 
(ref.)  104. 

Lecithoproteins,  309. 

Legler's  method  for  formaldehyde,   43. 

Lemon  oils  and  extracts,  determina- 
tion of  citral,  (ref.)  49. 

Leucosin,  339. 

Levulinic  acid  reaction,  58. 

Levulose,  51,  84,  85,  103,  105. 

Lieben's  test  for  alcohol,  5. 

Liebermann-Storch  reaction,   207. 

Liebig's  method  for  phosphorus,  303 ; 
for  sulphur,  296,  297. 

Light  filter,  93. 

Lignites,  253,  262. 

Lignoceric  acid,  134. 

Linoleic  acid,  136. 

Linolein,  146. 

Linolenic  acid,  137,  139. 

Linolenin,  146. 

Linseed  meal,  starch  content  of,  112. 

Linseed  oil,  141,  171,  203,  205,  (ref.) 
215-216 ;  vs.  paint  as  priming  coat, 
(ref.)  215. 

Lintner's  method  for  diastatic  power, 
115,  121. 

Lintner's  scale,  115,  116. 

Linusic  acid,  137. 


Low's  test  for  boric  acid,  374. 
Lubricants,  218,  222-235,  (ref.)  236-238. 
Lubricating  greases,  see  Lubricants. 
Lubricating  oils,  see  Lubricants. 
Lux-Ruhemann  test  for  saponifiable  oil, 
223. 

Mace,  starch  content  of,  112. 

Magnesium  nitrate  method  for  phos- 
phorus, 305. 

Mahler  calorimeter,  239. 

Maize  oil,  171,  182,  184,  199,  200,  204, 
208. 

Malic  acid  in  vinegar,  125. 

Malt  extracts,  114,  115. 

Maltosazone,  62,  64. 

Maltose,  52,  60,  70,  74,  84-86,  104-105. 

Malt  sugar,  see  Maltose. 

Malt  vinegar,  123. 

Mannose,  51,  85. 

Maple  sugar,  (ref.)  104. 

Maple  sirup,  (ref.)  104. 

Margarine,  197,  202. 

Marine  oils,  (ref.)  215.  (See  also 
Fish  oils.) 

Maumene  number,  144,  157,  159. 
(See  also  Fats.) 

Meat,  determination  of    starch  in,  112. 

Melissic  acid,  135. 

Melting  points,  6,  40,  64,  134-135, 
144,  167,  170,  180,  192,  193,  202. 

Menhaderi  oil,  171,  205. 

Metaformaldehyde,  37. 

Metaproteins,  309,  311. 

Methyl  alcohol,  24-26,  30-33. 

Methylene  blue  test  for  freshness  of 
milk,  (ref.)  367. 

Methylene-di-/3-Naphthol  test  for  for- 
maldehyde, 40. 

Methyl  ester  test  for  salicylic  acid,  382. 

Methylphenylhydrazine  as  reagent  for 
sugars,  63. 

Mett's  method  for  proteolytic  enzymes, 
325,  331-332. 

Milk,  349-368;  analysis  of,  352-368; 
composition  of,  349-352, 366-368;  con- 
densed, (ref.)  368;  detection  of  added 
water,  363,  364,  366-368;  of  cane 
sugar  and  calcium  sucrate,  (ref.) 
367-368:  of  heated,  367;  of  pre- 
servatives in,  365  (see  also  Food 
preservatives)  ;  determination  of 
dirt,  (ref.)  368 ;  homogenized,  (ref.) 
367 ;  preservation  of,  352,  366,  367 ; 
sampling,  352;  serum,  363,  366- 
368;  standards,  349,  364,  367; 
watered,  363,  366-368. 

Milk  chocolate,  (ref.)  104,  105,  367. 


SUBJECT   INDEX 


403 


Milk  sugar,  see  Lactose. 

Milk  supply,  (ref.)  366-368. 

Millon  reaction,  315,  382. 

Mineral   oil,    171,   206,   238,   263.     (See 

also  Lubricants,  Petroleum.) 
Mixed  oils,  viscosity  of,  229. 
Moisture  determination,  in  butter,  186  ; 

in    grain    products,    335 ;     in    milk, 

356  ;   in  sugar,  97. 
Molasses,      (ref.)      103-105;      as     fuel, 

(ref.)  262. 

Molisch  reaction  for  carbohydrates,  57. 
Monosaccharides,  50. 
Morley's  alcohol  table,  (ref.)  3. 
Morpurgo's  method  for  sucrol  or  dulcin, 

390. 

Mucic  acid  method,  59. 
Multirotation,  79,  86. 
Mulliken's  test  for  alcohol,  5. 
Munson    and    Walker    method    for    re- 
ducing sugars,  (ref.)  85. 
Mustard  oil,  171. 
Mutarotation,  79,  86. 
Muter's   method   for   fatty    acids,    136, 

138. 

Mutton  tallow,  170. 
Myricyl  alcohol,  169. 
Myristic  acid,  134,  195. 

Naphtha,  221. 

Naphthalene,  242. 

Neatsfoot  oil,  171. 

Neumann's  method  for  phosphorus,  304. 

New  method  for  diastatic  power,   117- 

120. 

New  scale  for  diastatic  power,  117-118. 
New  York  Board  of  Health  lactometer, 

354. 
New    York    Sugar    Trade    Laboratory, 

(ref.)  105. 
Nitrates,  in  food,   (ref.)   393;    in  milk, 

(ref.)   368;    nitrogen  in  presence   of, 

294. 

Nitric  acid  test  for  oils,  178. 
Nitrocompounds,  294. 
Nitrogen  determination,  288-295,  (ref.) 

306-307;      error    due    to    methane, 

(ref.)  307. 

Nitrogen-free  extract,  334. 
Nitrogenous  extractives,  312. 
Nucleoproteins,  309,  312. 
Nutmeg,  starch  content  of,  112. 

Oatmeal,  definition  and  standard,  343 ; 
starch  content  of,  112.  (For  methods 
of  analysis,  see  Grain  products.) 

Octodecyl  alcohol,  169. 

Oil,    almond,    171;     arachis,    171,    179, 


184,  (ref.)    199-202;    baobab,    (ref.) 
199-202 ;    castor,  142,  171 ;    coconut, 

170,  198,    (ref.)    199-202;     codliver, 

171,  (ref.)     199-202;      copac,     178; 
corn,   171,   182,   (ref.)   199-202;    cot- 
tonseed,   171,    177,   208,    (ref.)    199- 
202  ;  fish,  17,  208  ;  fish-liver,  171,  (ref.) 
199-202;    groups,   141;    hemp,   171; 
Japanese  sardine,  205 ;    kapok,   178, 
(ref.)  202;    lard,  171,  182,  184;    lin- 
seed, 171,  203,  (ref.)  214-217;  maize, 
171,   182,   184,  204,  208,   (ref.)   199- 
202;      menhaden,     171,     205,     208; 
mineral,    171,    206,    218-238;     mix- 
tures, 159,  213,  229 ;    mustard,  171 ; 
oleo,     171,     197;     olive,     171,    174- 

185,  (ref.)     199-202;      palm,     (ref.) 
199-202  ;  peanut,  see  Arachis ;  poppy- 
seed,   171,   182,   184,  204;    rapeseed, 

171,  184,  (ref.)  199-202;    rosin,  171, 
207;    salad,    see    Olive;    seal,    171; 
sesame,    171,    181,    184,    (ref.)    199- 
202;     soy   bean,    204;     sperm,    171; 
sunflower,     171;      tung,     171,     204; 
walnut,  204.     (See  also  Oils.) 

Oil  analysis,  140-185,  199-238. 

Oil  coatings,  preservative,  for  iron  and 
steel,  (ref.)  215. 

Oils,  acetyl  number,  160 ;  adulterants, 
213  (see  also  under  individual  oils) ; 
altered  by  age  or  oxidation,  210 ; 
analysis  (see  Oil  analysis)  ;  analytical 
properties,  table,  170-171 ;  blown, 
168 ;  bromine  absorption,  (ref.) 
215  ;  classification,  141 ;  commercial 
value,  214 ;  committee  report  on 
method  of  analysis,  (ref.)  173 ;  dry- 
ing, 141,  203-217;  edible,  174-202; 
fatty,  140-185,  199-217,  223-226; 
fish,  139,  205;  identification  of, 
213;  index  of  refraction,  164,  170- 
171 ;  iodine  number,  148-157,  170- 
172;  lubricating,  222-238;  Mau- 
mene  number,  157,  171;  "new  con- 
stant," (ref.)  173 ;  non-drying,  141  ; 
olive,  from  different  countries,  175- 
176;  oxidized,  210,  (ref.)  214-215; 
prices,  214;  refractometer  readings, 
164,  170-171,  172,  176,  211;  salad, 

172,  174,  (see  also  Oil,  olive) ;    semi- 
drying,    141 ;     specific   gravity,    162, 
171;     "unknown,"    213;     unsaponi- 
fiable  matter,    169,    173;    vegetable, 
drying,    203;     viscosity,    168,    226- 
231,  (ref.)  236-238. 

Oleic  acid,  135,  195,  197 ;  manufacture 
and  examination  of  commercial, 
(ref.)  139. 


404 


SUBJECT   INDEX 


Olein,  146. 

Oleomargarine,  197. 

Oleo  oil,  170,  197. 

Olive  oil,  141,  171,  174-185,  (ref.)  199- 
202,  216. 

Optical  activity,  of  carbohydrates,  78- 
85 ;  of  osazones,  63-64 ;  of  sugars, 
78-85  ;  of  vinegars,  125. 

Optical  method  for  proteolytic  enzymes, 
(ref.)  332,  333. 

Organic  compounds,  composition  and 
calorific  power,  245. 

Osazones,  62-69,  306. 

Osborne's  method  for  sulphur,  299. 

Ost  reagent,  78. 

Ostwald  pyknometer,  11. 

Oxidation,  of  alcohols,  23 ;  of  carbohy- 
drates, 58;  of  oils,  211. 

Oxygen  calorimeter,  168. 

Oxygen  method  for  sulphur,  301. 

Oxymethylene,  37. 

Paint,  203-205,  216,  217. 

Palmitic  acid,  134,  173,  195. 

Palmitin,  146,  173. 

Palm  oil,  170,  200. 

Parabrombenzylhydrazide  as  reagent 
for  sugars,  63. 

Paraffin,  170. 

"Paraffin  base"  petroleum,  221. 

Paraffin  oils,  water  in,  (ref.)  238. 

Paraformaldehyde,  37. 

Paraphenetol  carbamid,  390. 

Pavy's  reagent,  78. 

Peanut  oil,  171,  179. 

Peat,  (ref.)  263. 

Pensky-Martens  oil  tester,  233. 

Pentosans,  54-58,  61. 

Pentoses,  50,  57,  58. 

Pepper,  starch  content  of,  112. 

Pepsin,  324,  (ref.)  331-333. 

Peptids,  310. 

Peptones,  310,  311. 

Petroleum,  218-238,  247-251. 

Petroleum  oils,  calorific  powers,  248. 

Petroleum  production,  (ref.)  238. 

Phenylhydrazine  method  for  benzalde- 
hyde,  46. 

Phosphoproteins,  309,  312. 

Phosphorus  determination,  303-307. 

Phosphotungstic  acid  as  protein  pre- 
cipitant, 320. 

Phycetoleic  acid,  135. 

Phytosterol,  162,  172,  183,  194,  (ref.) 
200-201. 

Phytosterylacetate  test,  183,  194,  (ref.) 
200-201. 

Picric  acid  as  protein  precipitant,  322. 


Pine  wood  oils,  (ref.)  216. 

Platinum  resistance  thermometer,  (ref.) 
263. 

Polariscope,  80-94. 

Polariscopic  methods,  79-105. 

Polarization,  relation  to  sugar  content, 
94. 

Polarized  light,  78. 

Polenske  method,  199,  (ref.)  202. 

Polysaccharides,  50,  52. 

Poppyseed  oil,  171,  182,  184,  204,  215. 

Potassium  cyanide  method  for  formalde- 
hyde, 44. 

Potatoes,  determination  of  starch  in, 
106,  112,  (ref.)  121. 

Preservative  coatings  for  structural 
materials,  (ref.)  216. 

Preservatives  for  food,  see  Food  pre- 
servatives. 

Proof  spirit,  3,  4. 

Protamines,  309. 

Proteans,  309. 

Proteases,  323,  329,  331-333. 

Proteins,  308-323,  329-331 ;  coagulated, 
310  ;  conjugated,  309  ;  derived,  309  ; 
in  grain  products,  338 ;  in  milk,  369  ; 
simple,  308. 

Proteolytic  enzymes,  323-329,  331- 
333. 

Proteolytic  power,  324-329,  331-333. 

Proteoses,  310,  311. 

Pulfrich  refractometer,  165. 

Purity  of  sugar  solutions,  100. 

Pyknometer,  11,  164. 

Quartz  compensator,  81. 
Quartz  wedges,  81. 
Quevenne  lactometer,  353,  354. 
Quotient  of  purity,  100. 

Radiation  correction,  244. 
Rafinose,  50,  52,  59,  61,  85,  104. 
Rancidity  of  butter  fat,  194. 
Rapeseed  oil,  141,  171,  184,  202. 
Raw  sugar,  87,  104. 
Rectified  spirit,  4. 
Reducing  sugars,  69,  85,  86,  96. 
Redwood  viscosimeter,  227. 
Refraction,  index  of,  17-19,  32-33,  144, 

164,  166,  170-171,  172,  176,  211. 
Refractometer,       17-19,      32-33,       165, 

364. 
Refractometric  methods,    17-19,  32-33, 

165,  364,  367-368. 
Regnault-Pfaundler    cooling    correction, 

244. 

Regulations  of  international  commission 
on  sugar  analysis,  91. 


SUBJECT   INDEX 


405 


Reichert-Meissl  (Reichert-Wollny)  num- 
ber, 144,  148,  188,  196,  200-202.  * 

Renard-Tolman  method  for  arachidic 
acid,  179. 

Resin  acids  in  soap,  271,  287. 

Resin  and  resin  oil  in  linseed  oil,  207. 

Resinous  products  in  mineral  oils,  (ref.) 
237. 

Resorcin  test  for  formaldehyde,  38. 

Ricinoleic  acid,  138. 

Ricinolein,  146. 

Rose  method  for  fluorides,  375. 

Rosin,  170,  207;  in  shellac,  (ref.)  215; 
in  soaps,  271,  (ref.)  287 ;  in  varnishes, 
(ref.)  215 ;  size,  (ref.)  215. 

Rosin  oil,  171,  207. 

Rotation  and  rotating  power,   78-86. 

Rotation  dispersion,  83. 

Rubber,  determination  of  sulphur  in, 
(ref.)  307. 

Saccharimeter,  81,  82;  observations, 
unification  of,  104. 

Saccharin,  detection,  388,  (ref.)  391- 
395;  estimation  in  foods,  394. 

Saccharose,  see  Sucrose. 

Salad  oil,  174-185,  (ref.)  199-202. 

Salicylic  acid,  378-385,  (ref.)  391-395; 
action  of  ferric  chloride  on,  (ref.)  391 ; 
bromine  water  test,  382 ;  detection, 
378-385,  (ref.)  393,  395 ;  determina- 
tion, 378-384 ;  (ref.)  391-394  ;  ferric 
chloride  test,  delicacy,  interpreta- 
tion, 381 ;  in  pure  wines,  (ref.) 
391,  395;  in  strawberries,  (ref.) 
391 ;  in  fruits,  (ref.)  391  ;  Jorissen 
test,  382-383  ;  methyl  ester  test,  382  ; 
Millon's  reagent  test,  383  ;  reaction 
with  iron,  (ref.)  393 ;  substances 
mistaken  for,  in  ferric  chloride  test, 
381,  (ref.)  394,  395. 

Saliva  method  for  determination  of 
starch,  110. 

Saltpeter,  determination  in  meat,  (ref.) 
395. 

Sangle  Ferrieres  method  for  abrastol, 
390. 

Saponifiable  oil,  test  for,  in  lubricating 
oil,  223. 

Saponification,  data  on  pure  esters,  146  ; 
equivalent,  146  ;  number,  144  ;  num- 
ber of  butter  fat,  191,  196;  num- 
ber (table),  170-171. 

Sardine  oil,  Japanese,  205. 

Sativic  acid,  137. 

Sauer's  method  for  sulphur,  296. 

Sausages,  determination  of  starch  in,  112. 

Seal  oil,  171. 


Sesame,  meal,  influence  of  feeding,  on 
fat,  (ref.)  200;  oil,  171,  181,  184, 
(ref.)  199-202. 

Shellac  analysis,  (ref.)  216,  217. 

Sidersky  reagent,  78. 

Silicate  in  soap,  273. 

Sinibaldi's  method  for  abrastol,   389. 

Sitosterol,  172. 

Skeletins,  311. 

Smith  and  Menzies'  method  for  deter- 
mination of  boiling  point,  24. 

Soap,  266-275,-  (ref.)  286-287;  alumi- 
nate  in,  273;  borate  in,  273;  car- 
bonate in,  273  ;  chlorides,  271 ;  com- 
mercial, analysis,  266 ;  determina- 
tion of  water,  267 ;  extraction  with 
alcohol,  272 ;  fatty  acid  content, 
(ref.)  287;  fatty  and  resin  acids, 
269,  271;  free  alkali  or  acid,  273; 
glycerol  in,  270,  (ref.)  287 ;  industry, 
recent  development,  (ref.)  287 ; 
insoluble  matter,  274 ;  petroleum 
ether  extract,  268 ;  residue  insoluble 
in  alcohol,  273. 

Soaps,  antiseptic  value,  (ref.)  287; 
commercial,  composition,  (ref.)  287  ; 
free  alkali,  (ref.)  286 ;  from  different 
glycerides,  (ref.)  287 ;  germicidal 
and  insecticidal  values,  (ref.)  287  ; 
rosin  in,  287 ;  silicate,  273 ;  soluble 
fatty  acids,  270;  significance  in 
disinfectants,  (ref.)  287;  total,  free, 
and  carbonated  alkali,  (ref.)  286, 
287  ;  sugar  in,  270. 

Soldani  reagent,  78. 

Solidifying  points  of  fats,  167. 

Solids,  of  milk,  calculation  of,  355 ;  of 
milk,  determination  of,  356 ;  of 
vinegar,  124. 

Solution  densities  of  sugars,   (ref.)   105. 

Sorensen's  method  for  proteolytic  power, 
(ref.)  332. 

Soxhlet  lactometer,  353-354. 

Soy  bean  oil,  204. 

Specific  gravity,  11,  112,  133,  134,  162, 
170-171,  176,  185,  191,  196,  353, 
365. 

Specific  refractive  power,  211. 

Specific  rotating  (rotatory)  power,  82, 
83. 

Specific  temperature  reaction,  157 ;  of 
olive  oils  (table),  176. 

Spermaceti,  142,  170. 

Sperm  oil,  142,  171. 

Spices,  starch  content  of,  112. 

Spirit  vinegar,  124. 

Spontaneous  combustion,  test  for  oils, 
(ref.)  236. 


406 


SUBJECT   INDEX 


Sprigg's  method  for  peptic  activity, 
(ref.)  331. 

Standard  materials  for  calorimeter,  241. 

Standards,  alcohol,  2,  4,  29,  30 ;  butter, 
185 ;  grain  products,  343 ;  milk, 
364;  olive  oil,  177;  vinegar,  123. 

Starch,  52,  61;  soluble,  54,  55,  56; 
content  of  foods  and  spices,  112; 
determination  of,  106-113,  (ref.) 
121 ;  determination  in  cocoa  prod- 
ucts, (ref.)  121 ;  determination  in 
potatoes,  (ref.)  121 ;  hydrolysis  of, 
by  acid,  106 ;  hydrolysis  of,  by 
enzymes,  110,  113-120,  (ref.)  121- 
122;  rotating  power,  85;  sugar,  51. 

Steam-refined  cylinder  stock,  221. 

Stearic  acid,  134,  195 ;  glycerides  of, 
(ref.)  173. 

Stearin,  cottonseed,  170 ;  manufacture, 
methods,  (ref.)  173 ;  saponification 
number,  146. 

Straw,  starch  content  of,  112. 

Stutzer  method  for  protein  nitrogen, 
(ref.)  329  ;  reagent,  317. 

Sucrol,  390. 

Sucrose,  51 ;  Clerget  method  for,  94 ; 
combustion,  241;  determination  by 
use  of  invertase,  (ref.)  105 ;  determi- 
nation in  beets  and  cane,  98 ;  deter- 
mination in  condensed  milk,  (ref.) 
103 ;  determination  in  milk,  (ref.) 
367,  368  ;  determination  in  molasses, 
(ref.)  105 ;  determination  in  presence 
of  commercial  glucose  (ref.)  103 ; 
hydrolysis,  60;  hydrolysis,  (ref.)  104; 
rotating  power,  84,  85 ;  solubility  in 
alcohol,  55. 

Sugar,  see  also  Sucrose ;  analysis,  87 ; 
beet,  optically  active  non-sugars  of, 
(ref.)  105;  beets,  analysis  of,  98, 
(ref.)  104,  105  ;  beets,  see  also  Beets ; 
cane,  determination  of  sucrose  in, 
98 ;  in  soap,  270 ;  manufacture  con- 
trol, (ref.)  104,  105;  mixtures,  analy- 
sis of,  101,  102  ;  mixtures,  analysis  of, 
(ref.),  85,  86;  periodicals  devoted 
to,  103 ;  solutions,  density,  and  pu- 
rity of,  100;  vinegar,  124,  126,  127. 

Sugars,  50-105 ;  behavior  on  oxidation, 
58;  toward  caustic  alkalies,  (ref.), 
86;  toward  Fehling's  solution,  69, 
(ref.)  86 ;  toward  hydrazines,  61 ; 
estimation  by  means  of  refractom- 
eter,  101,  (ref.)  104,  105;  identifi- 
cation, 101 ;  oxidation  of,  (ref.) 
86;  rotatory  powers  of,  84,  85; 
"  unknown,"  101.  (See  also  Car- 
bohydrates.) 


Sulphite  method  for  benzaldehyde,  46 ; 
sodium,  recoverable  from  food,  (ref.) 
392.  (See  also  Sulphites.) 

Sulphites,  determination  in  foods,  (ref.) 
393;  in  gelatin,  (ref.)  392,  393, 
394;  in  green  corn,  (ref.)  393; 
in  lime  juice,  (ref.)  394 ;  in  meat, 
(ref.)  392,  395;  in  molasses,  (ref.) 
394;  in  sugar  products,  (ref.)  392. 
(See  also  Sulphurous  acid.) 

Sulphur,  295-303 ;  compounds,  volatile 
in  meat  and  influence  on  detection 
of  added  sulphites,  (ref.)  393;  de- 
termination, 295-303,  (ref.)  306-307 ; 
determination  in  petroleum,  237; 
in  coal,  257,  26-2,  264. 

Sulphurous  acid,  377  ;  behavior  in  foods, 
(ref.)  392 ;  detection  in  foods,  (ref.) 
391 ;  detection,  377 ;  disappearance 
when  added  to  wine,  (ref.)  395 ; 
organically  combined  in  foods,  (ref.) 
391 ;  quantitative  estimation,  377. 

Sunflower  oil,  171. 

Suspended  matter  in  oils,  235. 

Suzzi,  notes  on  Maumene  number,  160. 

Tallow,  beef,  170;  detection  of  lard, 
(ref.)  201 ;  group,  141 ;  mutton,  170. 

Tan  bark  as  boiler  fuel,  (ref.)  264. 

Tannin,  as  protein  precipitant,  321. 

Tar  oils,  (ref.)  217. 

Temperature  coefficients  in  polarization 
of  raw  sugars,  (ref.)  104. 

Thomas  and  Weber's  method  for  pro- 
teolytic  power,  (ref.)  331. 

Titer  test,  144,  167. 

Tocher's  test,  182. 

Tollen's  aldehyde  reagent,  34,  35. 

Total  solids,  determination  in  milk,  356. 

Treasury  Dept.  methods  of  sugar  analy- 
sis, (ref.)  104. 

Triazo  nitrogen,  determination  of,  (ref.) 
307. 

Trichloracetic  acid,  as  protein  precipi- 
tant, 322. 

Triglycerides,  in  butter  fat,  195 ;  mixed, 
143;  simple,  143. 

Trioxy  methylene,  37. 

Trisaccharide,  50,  52. 

Tryptic  activity,  325. 

Tryptophan  reaction,  315. 

Tung  oil,  171,  204. 

Turmeric  test  for  boric  acid  or  borates, 
374;  Low's  modification,  374. 

Turpentine,  (ref.)  215-216  ;  adulterants, 
(ref.)  215,  217;  benzene  in,  (ref.) 
216,  217;  commercial,  of  United 
States,  (ref.)  217 ;  naphtha  in,  (ref.) 


SUBJECT    INDEX 


407 


216;    oils,  analysis,  (ref.)  216;    sub- 
stitutes, (ref.)   215-217;    wood,  pro- 
duction, etc.,  (ref.)  217. 
Twitchell's  Method  for  fatty  acids,  271. 

Unification,  of  methods  for  diastatic 
power,  120 ;  of  reducing  sugar 
methods,  (ref.)  85 ;  of  saccharimeter 
observations,  (ref.)  104. 

Unsaponifiable  matter  of  fats  and  waxes, 
169,  (ref.)  173,  237. 

Unsaponifiable  oils,  determination  of,  in 
lubricants,  224. 

Vanilla  extract,  analysis  of,  (ref.)  49. 

Vanillin,  determination,  48,  49. 

Van  Slyke's  method  for  analysis  of 
proteins,  (ref.)  331. 

Vegetable  drying  oils,  203. 

Ventzke  scale,  82,  88. 

Vinegar,  123-132,  (ref.)  131,  132; 
analyses  of  typical,  126 ;  determina- 
tion of  source,  124 ;  glycerol  con- 
tent of,  (ref.)  132 ;  identification  of, 
124;  methods  of  analysis,  127; 
standards,  123,  132. 

Viscosimeter,  226. 

Viscosity,  168,  226,  (ref.)  236-238;  of 
fats,  144 ;  of  illuminating  oils,  (ref.) 
237  ;  numbers,  184  ;  test,  Abraham's 
modification,  183 ;  test,  Blasdale's, 
183. 

Volatile  matter,  average  relation  to 
calorific  power  (table),  260;  in  coke 
and  anthracite,  (ref.)  264 ;  of  coal, 
256. 

Volhard's  method  for  proteolytic  power, 
(ref.)  332. 

Walker's  rule,  246. 

Walnut,  oil,  204 ;  shells,  starch  content 
of,  112. 

Washing  powders,  disinfectant  prop- 
erties, (ref.)  286. 


Water  determination,  see  Moisture. 
Water,   equivalent  of  calorimeter,   241 ; 

density  of,  17  ;   in  butter,  186. 
Watering  of  milk,  detection  of,  363,  364. 
Wax,   bees,    170 ;     carnatiba,    170,    173 ; 

Japan,  170. 
Waxes,     alcohols    of,     169 ;      analytical 

properties  (table),  170-171 ;    classifi- 
cation,  141 ;    general  methods,   140 ; 

Unsaponifiable  matter  of,  169. 
Welman's  reaction,  (ref.)  201. 
Welter's  rule,  246. 
Westphal  balance,  162. 
Whale  oil,  142,  171. 
Wheat,  see  Grain  products. 
Whisky,  (ref.)  32,  33. 
Wijs    method    and    solution,    152,    156, 

(ref.)  173. 
Wiley's  method  for  melting  point,  192, 

202. 

Wilfarth  method  for  nitrogen,  389. 
Wine  vinegar,  123. 
Wohlgemuth's     method     for     diastatic 

power,    116,    (ref.)    122;    scale,    117, 

(ref.)  122. 
Wood,  calorific  power,  253  ;  oil,  Chinese, 

204;     oils,    Philippine,    (ref.),    215; 

ultimate     composition,     252 ;      and 

similar  fuels,  251. 
Wood  alcohol,  requirements  for  use  in 

denaturing,  30. 
Wright's    factors    for    specific    gravity 

calculations,  164. 

Xanthoproteic  reaction,  315. 
Xylosazone,  62,  63,  64. 
Xylose,  63,  85. 

Yeasts,     pure,     separation     of     carbo- 
hydrates by,  104. 

Zeiss  refractometer,  165. 

Zero-point  of  polariscope,  80,  81,  87. 

Zinc  sulphate,  as  protein  precipitant,  316. 


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